PICHIA PASTORIS STRAINS FOR PRODUCING PREDOMINANTLY

HOMOGENEOUS GLYCAN STRUCTURE

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority from U.S. Provisional Application No. 61/717,423, filed October 23, 2012, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Pichia pastoris is a highly successful system for production of a wide variety of recombinant proteins. Several factors have contributed to its rapid acceptance, including: (1) a promoter derived from the alcohol oxidase I (AOX1) gene of P. pastoris that is uniquely suited for the controlled expression of foreign genes; (2) the similarity of techniques needed for the molecular genetic manipulation of P. pastoris to those of Sacchawmyces cerevisiae; and (3) the strong preference of P. pastoris for respiratory growth, a physiological trait that facilitates its culturing at high-cell densities relative to fermentative yeasts.

[0003] As a yeast, P. pastoris is a single-celled microorganism that is easy to manipulate and culture. However, it is also a eukaryote and capable of many of the post-translational modifications performed by higher eukaryotic cells such as proteolytic processing, folding, disulfide bond formation and glycosylation. Thus, many proteins that would end up as inactive inclusion bodies in bacterial systems are produced as biologically active molecules in P. pastoris. The P. pastoris system is also generally regarded as being faster, easier, and less expensive to use than expression systems derived from higher eukaryotes such as insect and mammalian tissue culture cell systems and usually gives higher expression levels.

[0004] P. pastoris has the potential of performing many of the posttranslational

modifications typically associated with higher eukaryotes. These include processing of signal sequences (both pre- and prepro-type), folding, disulfide bridge formation, and both O- and N-linked glycosylation. Glycosylation of secreted foreign (higher) eukaryotic proteins by P. pastoris and other fungi can be problematic. In mammals, O-linked oligosaccharides are composed of a variety of sugars including N-acetylgalactosamine, galactose and sialic acid. In contrast, lower eukaryotes, including P. pastoris, may add O-oligosaccharides solely composed of mannose (Man) residues.

[0005] N-glycosylation in P. pastoris is also different than in higher eukaryotes. In all eukaryotes, it begins in the ER with the transfer of a lipid-linked oligosaccharide unit, Glc3Man9GlcNAc2 (Glc = glucose; GlcNAc = N-acetylglucosamine), to asparagine at the recognition sequence Asn-X-Ser/Thr. This oligosaccharide core unit is subsequently trimmed to Man8GlcNAc2. It is at this point that lower and higher eukaryotic glycosylation patterns begin to differ. The mammalian Golgi apparatus performs a series of trimming and addition reactions that generate oligosaccharides composed of either Man5-6GlcNAc2 (high- mannose type), a mixture of several different sugars (complex type) or a combination of both (hybrid type). Two distinct patterns of N-glycosylation have been observed on foreign proteins secreted by P. pastoris. Some proteins are secreted with carbohydrate structures similar in size and structure to the core unit (Man8-11G1CNAC2). Other foreign proteins secreted from P. pastoris receive much more carbohydrate and appear to be

hyperglycosylated.

[0006] N-linked high mannose oligosaccharides added to proteins by yeasts represent a problem in the use of foreign secreted proteins by the pharmaceutical industry. For example, they can be exceedingly antigenic when introduced intravenously into mammals and furthermore may cause rapid clearance of the protein from the blood by the liver.

[0007] In an attempt to modify the N-glycosylation pathway of Pichia pastoris, a strain (hereinafter referred to as "M5-Blast") was created, as described in Jacobs et al., 2009, Nature Protocols 4:58-70. M5-Blast is a modification of the P. pastoris GS115 strain wherein the endogenous mannosyltransferase gene OCHl is disrupted by the introduction of a cassette comprising an a- 1,2 mannosidase gene. However, the M5-Blast strain is subject to genomic rearrangements that regenerate the endogenous OCHl gene and in parallel remove the a- 1,2 mannosidase gene after rounds of freezing and thawing, growth under various temperatures and conditions, and from subsequent transformations with other plasmids to introduce exogenous genes. [0008] SUMMARY OF THE DISCLOSURE

[0009] Disclosed herein are novel Pichia pastoris strains for expression of exogenous proteins with substantially homogeneous N-glycans. More specifically, the strains are genetically engineered to include a mutant OCHl allele which is transcribed into an mRNA coding for a mutant OCHl gene product (i.e., -l,6-mannosyltransferase, or "OCHl protein"). The mutant OCHlprotein contains a catalytic domain substantially identical to that of the wild type OCHl protein, but has an N-terminal sequence that alters the localization of the OCHl protein to or in the Golgi apparatus. The strains do not include any other OCHl allele that produces an mRNA coding for a functional OCHl protein. Such strains are robust, stable, and transformable, and the mutant OCHl allele and the associated phenotype (i.e., ability to produce substantially homogeneous N-glycans) are maintained for generations, after rounds of freezing and thawing, and after subsequent transformations.

[0010] This disclosure also features methods of constructing the strains, as well as methods of expressing proteins via the strains.

[0011] Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1. Diagram of deletion strategy for removing the OCHl N-terminal fragment homology from M5-Blast Pichia genome. Homology arms flanking the OCHl N- terminal fragment are used to create a double crossover construct containing lox71-lox66 recombination sites. The intervening sequences can be removed by ere mediated

recombination.

[0013] Figure 2. PC primers for amplification of flanking arms of double crossover construct from M5-Blast genomic DNA.

[0014] Figure 3. PCR reactions for the addition of lox sites to the ends of the homology arms. [0015] Figure 4. Addition of M5-Blast genomic DNA extensions onto existing loxll- Λί<·.<; ' Ναι^ !ox0 cassette.

[0016] Figure 5. Overlap assembly and amplification of the final sequence for generating the double crossover fragment for M5-Blast Pichia transformation. This final construct has >5Q0 bp of homology arms flanking the selection/counter-selection cassette.

[0017] Figure 6. PGR primer pairs used to generate DNA fragment for double crossover recombination event.

[0018] Figure 7. Theoretical arrangement of LEi/5---mannosidaseHDEL region of the M5- Blast genome after double crossover recombination event.

[0019] Figure 8. Theoretical arrangement of genomic DNA between LEU 5 and

mannosidaseHDEL after ere recombination.

[0020] Figure 9. PC primers 81487118-80765854 used to verify DNA sequence of region that could have been derived from PCR products transformed into the M5-Blast strain.

[0021] Figure 10. N-glycan analysis of a recombinant protein expressed in various P.

pastoris strains.

[0022] Figure 11. N-glycan analysis of trastuzumab obtained in Study 1 described in Example 6.

[0023] Figure 12. Comparison of Her2 binding affinity of Man5-type trastuzumab (Study 1) and commercial Herceptin by ELISA.

[0024] Figure 13. DSA-FACE analysis of the total N-glycan pool on medium proteins from 'Trans' strains. A. result for a malto-dextrose reference. Panel B to F show results for N-glycans, as follows: B. GS Trans strain in microscale; C. M5 Trans strain in microscale; D. GS Trans strain in bioreactor; E. M5 Trans strain in bioreactor; F. reference N-glycans from bovine RNase B.

[0025] Figure 14. DSA-FACE analysis of the total N-glycan pool on medium proteins from 'CalB' strains. A. result for a malto-dextrose reference. Panel B to F show results for N-glycans, as follows: B. GS CalB strain in microscale; C. M5 CalB strain in microscale; D. GS CalB strain in bioreactor; E. M5 CalB strain in bioreactor; F. reference N-glycans from bovine RNase B.

[0026] Figure 15. DSA-FACE analysis of the total N-glycan pool on medium proteins from 'CalA' strains. A. result for a malto-dextrose reference. Panel B to F show results for N-glycans, as follows: B. GS CalA strain in microscale; C. M5 CalA strain in microscale; D. GS CalA strain in bioreactor; E. M5 CalA strain in bioreactor; F. reference N-glycans from bovine RNase B.

[0027] Tables 1-6 are set forth on pages 36-49. Tables 7-8 are found on page 32 (Example 7).

[0028] Table 1 lists the DNA sequence (SEQ ID NO: 1) of the OCH1 locus in a SuperM5 strain described in Example 1.

[0029] Table 2 lists the amino acid sequence for wild type OCH1 (SEQ ID NO: 2) in Pichia pastoris.

[0030] Table 3 lists nucleotides that may be deleted from the Upstream OCH1 segment.

[0031] Table 4 lists the DNA sequence for the OCH1 locus (+/- 2kb) for the M5-Blast Pichia pastoris strain.

[0032] Table 5 lists the amino acid sequence and nucleotide sequence for the Upstream OCH1 segment.

[0033] Table 6 lists the amino acid sequence and nucleotide sequence for the Downstream OCH1 segment.

[0034] Table 7. N-glycan analysis of trastuzumab obtained from Study 2 (Example 6). [0035] Table 8. Kinetic parameters of trastuzumab analyzed on BIAcore (Example 6). DETAILED DESCRIPTION

[0036] Genetically engineered Pichia pastoris strains

[0037] This disclosure features novel genetically engineered Pichia pastoris strains which are robust, stable, and transformable, and which produce proteins with substantially homogeneous N-glycan structures.

[0038] As further described herein, the strains are genetically engineered to include a mutant OCHl allele which is transcribed into an mRNA coding for a mutant OCHl gene product (i.e., -l,6-mannosyltransferase, or "OCHl protein"). The mutant OCHlprotein contains a catalytic domain substantially identical to that of the wild type OCHl protein, but has an N-terminal sequence that alters the localization of the OCHl protein to or in the Golgi apparatus. The strains do not include any other OCHl allele that produces an mRNA coding for a functional OCHl protein.

[0039] The strains can be additionally genetically engineered to contain a nucleic acid coding for and expressing an cc-l,2-mannosidase which converts the M8 N-glycan,

Man8GlcNAc2, to the M5 N-glycan, Man5GlcNAc2.

[0040] As a result of the genetic modifications, the strains disclosed herein produce substantially homogeneous N-glycans.

[0041] By "substantially homogeneous" N-glycans it is meant that given a preparation containing a population of a particular glycoprotein of interest, at least 50%, 60%, 75%, 80%, 85%, 90% or even 95% of the N-glycans on the protein molecules within the population are the same.

[0042] By "predominant N-glycan structure" or "predominant glycoform" it is meant a specific N-glycan structure or glycoform of (i.e., attached to) a protein constitutes the greatest percentage of all N-glycan structures or glycoforms of the protein. In certain specific embodiments, a predominant glycoform accounts for at least 40%, 50%, 60%, 70%, 80%, 90% or 95% or greater of the population of all glycoforms on the protein. Examples of desirable N-glycan structures include, e.g., Man8GlcNAc2 (or "M8") or

Man5GlcNAc2("M5"). Additional desirable N-glycan structures include, GnM5

(GlcNAcMan ₅ GlcNAc ₂ ), GalGnM5 (GalGlcNAcMan ₅ GlcNAc ₂ ), GalGnM3

(GalGlcNAcMan ₃ GlcNAc ₂ ), GnM3 (GlcNAcMan ₃ GlcNAc ₂ ), Gn2M3

(GlcNAc ₂ Man ₃ GlcNAc ₂ ), and Gal2Gn2M3 (Gal ₂ GlcNAc ₂ Man ₃ GlcNAc ₂ ). The structures of these N-glycans have been described, e.g., in Jacobs et al., 2009, Nature Protocols 4:58-70, incorporated herein by reference.

[0043] In a specific embodiment, the strains of this invention include both a mutant OCHl allele and a nucleic acid coding for and expressing an cc-l,2-mannosidase, such that the strains produce homogeneous N-glycans with M5 being the predominant glycoform. These strains are also referred to herein as SuperM5 or SuperMan5 strains. An example of a SuperM5 strain is described in the Example section below.

[0044] The strains of this invention are "robust", which means that the strains (unless noted otherwise as an auxotroph or deficient strain, e.g., protease deficient, AOX1 mutant, etc.) have approximately the same growth rate and the same growth conditions as unmodified Pichia pastoris strains such as strain GS115. For example, the strains of this invention can grow at elevated temperatures (e.g., 30°C, 37°C or even 42°C) and are not temperature sensitive. For example, the SuperM5 strains disclosed herein are robust and are not temperature sensitive.

[0045] The strains of this invention are also stable, which means that the genetic

modifications and the phenotype as a result of the genetic modifications (i.e., producing homogeneous N-glycans) are maintained through generations, e.g., at least 10, 20, 30, 40 or 50 generations (cell divisions), after rounds of freezing and thawing, and after subsequent transformations. For example, the SuperM5 strains disclosed herein maintain the mutant OCHl allele through generations and are able to continue making substantially homogeneous M8 (or other downstream N-glycans), without reversion..

[0046] Genetic engineering - mutant OCHl allele

[0047] The strains of this invention are genetically engineered to include a mutant OCHl allele which is transcribed into an mRNA coding for a mutant OCHl gene product (i.e., cc- 1,6-mannosyltransferase, or the "OCHl protein"). The mutant OCHl protein contains a catalytic domain substantially identical to that of the wild type OCHl protein but has an N- terminal sequence that alters the localization of the OCHl protein to or in the Golgi apparatus.

[0048] The wild type OCHl gene of Pichia pastoris has an open reading frame that encodes a protein of 404 amino acids (SEQ ID NO: 2). Like other fungal Golgi glycosyltransferases, the Pichia pastoris OCHl protein is a type II membrane protein, has a short cytoplasmic tail (Metl to Tyr21 (SEQ ID NO: 25), or Ala2 to Tyr21), a membrane anchor domain (Phe22 to Ser44, i.e., FYMAIFAVSVICVLYGPSQQLSS (SEQ ID NO: 89)), a stem region, and a large C-terminal region containing the catalytic domain. See, e.g., Kim et al., J. Biol. Chem.

281:6261-6272 (2006); Nakayama et al., EMBO 11(7): 2511-2519 (1992); and Tu et al., Cell. Mol. Life Set 67:29-41 (2010).

[0049] The wild type OCH1 protein is generally localized in cis-Golgi. Golgi localization of the wild type OCH1 protein is believed to be dictated by the N-terminal region consisting of the cytoplasmic tail, the membrane anchor domain, and the stem region. In particular, the membrane anchor domain, including its amino acid constituents and length, plays an important role in the Golgi targeting of the protein. See, e.g., Tu et al. {supra).

[0050] The mutant OCH1 protein of this disclosure has an N-terminal sequence that alters the Golgi localization of the mutant OCH1 protein, as compared to the wild type OCH1 protein. As a result of this altered N-terminal sequence, the mutant OCH1 protein is either not properly targeted to or retained within the Golgi apparatus, or not properly targeted to or retained within the correct compartment within Golgi. The term "targeting" is meant the biological mechanisms by which proteins are transported to the appropriate destinations in the cell or outside of the cell. In specific embodiments, the mutant OCH1 protein of this disclosure lacks an N-terminal sequence that allows the Golgi targeting of the mutant OCH1 protein, such that the mutant OCH1 protein is not targeted the Golgi apparatus and is transported to another cellular location or secreted to outside of the cell.

[0051] In some embodiments, the alteration in the N-terminal sequence is a result of a mutation, i.e., addition, deletion or substitution, of one or more amino acids in the membrane anchor domain of the OCH1 protein. In specific embodiments, one or more amino acids in the membrane anchor domain have been deleted. In particular embodiments, at least 2, 3, 4, 5, 6, 7 or more amino acids, contiguous or otherwise, of the membrane anchor domain have been deleted. For example, some or all of the first 5 amino acids (FYMAI, SEQ ID NO: 90) of the membrane anchor domain are deleted.

[0052] In other embodiments, the alteration in the N-terminal sequence is a result of a mutation, i.e., addition, deletion or substitution, of one or more amino acids in the

cytoplasmic tail of the OCH1 protein. In specific embodiments, one or more amino acids in the cytoplasmic tail have been deleted; for example, at least 2, 3, 4, 5, 6, 7 or more amino acids, contiguous or otherwise, of the cytoplasmic tail have been deleted. Examples of deletions in the cytoplasmic tail are found in Table 3. In other embodiments, deletion of one or more amino acids is combined with addition of one or more amino acids in the cytoplasmic tail.

[0053] In still other embodiments, the alteration in the N-terminal sequence is a result of a mutation of one or more amino acids in the stem region of the OCHl protein; for example a deletion of one or more amino acids in the first 10, 20, 30, 40, 50, or 60 amino acids immediately following the membrane anchor domain.

[0054] In certain embodiments, the alteration in the N-terminal sequence is a result of a combination of mutations in the cytoplasmic tail, the membrane anchor domain, and/or the stem region of the OCHl protein.

[0055] In specific embodiments, the alteration in the N-terminal sequence is a result of a combination of mutations in the cytoplasmic tail and the membrane anchor domain. For example, one or more amino acids in the cytoplasmic tail and one or more amino acids in the membrane anchor domain have been deleted. Examples of deletions in the N-terminal region of the OCHl protein are listed in Table 3.

[0056] In other embodiments, in addition to deletions in one or more domains, one or more amino acids are added to the N-terminus of the protein, as long as the resulting mutant N- terminal sequence still fails to properly target or localize the OCHl protein in Golgi. For example, the resulting mutant N-terminal sequence still lacks a functional membrane anchor domain. Whether a mutant sequence includes a membrane anchor domain can be readily determined based on the amino acid compositions and length. The membrane anchor domain of Golgi glycosyltransferases typically consists of 16-20 amino acids, which are hydrophobic and often contain aromatic amino acids, and has hydrophilic, often positively charged amino acids immediately outside both ends of the membrane span. See, e.g., Nakayama et al.

(1992), supra. One example of a mutant OCHl protein is set forth in SEQ ID NO: 3, which has its first 10 amino acids in place of the first 26 amino acids of the wild type OCHl protein.

[0057] The mutant OCHl protein disclosed herein contains a catalytic domain substantially identical to that of the wild type OCHl protein.

[0058] The catalytic domain of the wild type OCHl protein is located within the C-terminal fragment of 360 amino acids (i.e., within amino acids 45 to 404 of SEQ ID NO: 2). In some embodiments, the mutant OCHl protein comprises a C-terminal fragment that is substantially identical to amino acids 45-404, 55-404, 65-404, 75-404, 85-404, 95-404, or 105-404 of SEQ ID NO: 2. By "substantially identical" it is meant that the sequences, when aligned in their full lengths, are at least 90%, 95%, 98%, 99%, or greater, identical. In most embodiments, the catalytic domain of the mutant OCHl protein does not differ from the wild type domain by more than 10 amino acids, 8 amino acids, 5 amino acids, 3 amino acids, or 2 amino acids. In specific embodiments, the catalytic domain of the mutant OCHl protein is identical with that of the wild type OCHl protein. When one or more amino acids are different, it is preferable that the differences represent conservative amino acid substitutions. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as I, V, L or M for another; the substitution of one polar (hydrophilic) residue for another polar residue, such as R for K, Q for N, G for S, or vice versa; and the substitution of a basic residue such as K, R or H for another or the substitution of one acidic residue such as D or E for another.

[0059] The mutant OCHl protein also substantially retains the catalytic activity of the wild type OCHl protein, i.e., at least about 75%, 80%, 85%, 90%, 95% or more, of the a- 1,6- mannosyltransferase activity of the wild type OCHl protein. The activity of a particular OCHl mutant protein can also be readily determined using in vitro or in vivo assays known in the art. See, e.g., Nakayama (1992), supra.

[0060] As described above, the strains of this invention include a mutant OCHl allele which is transcribed into an mRNA coding for a mutant OCHl protein, and do not include any other OCHl allele that produces an mRNA coding for a functional OCHl protein. Such strains can be engineered by a variety of means.

[0061] In some embodiments, the wild type OCHl allele at the OCHl locus on the chromosome of a Pichia pastoris strain has been modified or mutated to provide a mutant OCHl allele (as illustrated in the Examples hereinbelow), or has been replaced by a mutant OCHl allele (e.g., through homologous recombination). The modifications should be such that the resulting strain is stable with respect to the mutant OCHl allele. That is, the mutant allele is maintained in the strain through generations (e.g., at least 10, 20, 30, 40, 50 or more cell divisions) suitable for both small volume flask culture and industrial size bioreactor culture, without reverting to an OCHl allele coding for a functional OCHl protein.

[0062] In other embodiments, a mutant OCHl allele is introduced through an expression vector into a Pichia pastoris strain whose wild type OCHl allele(s) (wild type OCHl "allele" if haploid, or wild type OCHl "alleles" if diploid) has already been disrupted hence no functional OCHl protein is produced from the native OCHl allele or native OCHl locus. The expression vector can be an integrative vector designed to integrate the mutant OCHl allele into the host genome; or a replicative vector (e.g., a plasmid) which replicates in the strain independent of the chromosomes.

[0063] Whether the mutant OCHl allele is created at the native OCHl locus by mutating or replacing the wild type OCHl allele, or is provided via an expression vector in a strain whose wild type OCHl allele(s) (wild type OCHl "allele" if haploid, or wild type OCHl "alleles" if diploid) has already been disrupted, it is important that the resulting mutant strain does not produce functional OCHl protein through generations (e.g., at least 10, 20, 30, 40, 50 or more cell divisions). By "functional OCHl protein" it is meant the wild type OCHl protein or a functional equivalent of the wild type OCHl protein, i.e., a protein that is targeted to Golgi and substantially retains the catalytic activity of the wild type OCHl protein (i.e., at least about 80%, 85%, 90%, 95% or more, of the -l,6-mannosyltransferase activity of the wild type OCHl protein). To avoid reversion, homologous sequences in the strain should be removed to avoid homologous recombination which generates a wild type OCHl allele.

[0064] The mutant OCHl allele, whether present on the host chromosome or on an extra- chromosomal vector, is transcribed into mRNA. In other words, the strain is engineered such that the coding sequence of the mutant OCHl allele is operably linked to a promoter to effect transcription. The promoter can be an endogenous promoter, such as the endogenous OCHl promoter, a promoter heterologous to the OCHl allele (e.g., an AOX1 promoter, a GAP promoter), and the like; or can be an exogenous promoter functional in Pichia pastoris. The level of transcription can be the same as, higher or lower than, the level of transcription of the wild type OCHl allele in an unmodified Pichia pastoris strain (such as GS115).

[0065] Pichia pastoris strains having the genetic modifications to the OCHl allele(s) described above include both haploid strains and diploid strains. For diploid strains having an OCHl mutant allele integrated into a host chromosome, the strains can be either homozygous or heterozygous for the OCHl mutant allele.

[0066] Pichia pastoris strains having the genetic modifications to the OCHl allele(s) described above are robust and stable, and produce proteins with substantially homogeneous N-glycan structures with Man8GlcNAc2 being the predominant N-glycan.

[0067] Genetic engineering - A nucleic acid coding for and expressing an oc-1,2- mannosidase

[0068] In addition to the genetic modifications to the OCH1 allele(s) described above, the strains can be engineered to include a nucleic acid molecule which codes for and is capable of expressing an cc-l,2-mannosidase or a functional fragment thereof which converts

Man ₈ GlcNA _c2 to Man ₅ GlcNA _c2 , thereby providing Man ₅ GlcNA _c2 as the predominant N- glycan form.

[0069] cc-l,2-mannosidase (MS-I) is a well characterized family of enzymes. Most MS-I enzymes are known to be localized in the Golgi or endoplasmic reticulum, although a few are secreted and have extracellular activity. See, Gonzalez et al., Mol Biol Evolution 17:292-300 (2000). The topology of those enzymes that localize to the ER and the Golgi generally includes a luminal catalytic domain and an N-terminal transmembrane region. See,

Herscovics, Biochimie 8: 757-62 (2001). The N-terminal region is composed of a stem region (closest to the luminal catalytic domain), a transmembrane domain, and a cytoplasmic tail. In the secreted MS-I enzymes, the extra-catalytic transmembrane region is also known as a leader sequence, serving as a signal for secretion of the enzyme. Detailed

characterizations of various cc-l,2-mannosidases can be found in Becker et al. (European J. Cell Biol 79: 986-992 (2000)) which studied the MS-I enzymes from mouse and S. cerevisiae and their catalytic domains; Schneikert and Herscovics (Glycobiology 4: 445-450 (1994)) which characterized the catalytic activity of a murine MS-I and its catalytic domain;

Gonzalez et al. (J. Biol Chem 274: 21375-86 (1999)) which examined the activities and domains of several MS-I enzymes, including two enzymes from C. elegans, a human MS-I and the S. cerevisiae MS-I (from the ER); and Maras et al. (J. Biotechnology 77:255-263 (2000)), which characterizes the T. reesei cc-l,2-mannosidase as belonging to the category of secretory MS-I's, which are composed of a catalytic domain and an N-terminal leader sequence.

[0070] The nucleic acid molecule encoding an cc-l,2-mannosidase or a functional fragment thereof can derive from any species for use in this invention, including but not limited to mammalian genes encoding, e.g., murine cc-l,2-mannosidase (Herscovics et al. J. Biol. Chem. 269: 9864-9871, 1994), rabbit cc-l,2-mannosidase (Lai et al. J. Biol. Chem. 269: 9872-9881, 1994), or human cc-l,2-mannosidase (Tremblay et al. Glycobiology 8: 585-595, 1998), fungal genes encoding, e.g., Aspergillus cc-l,2-mannosidase (msdS gene), Trichoderma reesei CC-1,2- mannosidase (Maras et al., J. Biotechnol. 77: 255-263, 2000), or a Saccharomyces cerevisiae CC-l,2-mannosidase, as well as other genes such as those from C. elegans (GenBank

Accession Nos. CAA98114 and CAB01415) and Drosophila melanogaster (GenBank Accession No. AAF46570) (see, e.g., Nett et al., Yeast 28:237-252, 2011, incorporated herein by reference).

[0071] By "functional part" or "enzymatically active fragment" of an cc-l,2-mannosidase, it is meant a polypeptide fragment of a naturally occurring or wild type cc-l,2-mannosidase which substantially retains the enzymatic activity of the full-length protein. By "substantially" in this context it is meant at least about 75%, 80%, 85%, 90%, 95% or more, of the enzymatic activity of the full-length protein is retained. For example, the catalytic domain of an cc-1,2- mannosidase, absent of any N-terminal transmembrane or signal sequence, constitutes a "functional fragment" of the cc-l,2-mannosidase. Those skilled in the art can readily identify and make functional fragments of an cc-l,2-mannosidase based on information available in the art and a combination of techniques known in the art. The activity of a particular polypeptide fragment can also be verified using in vitro or in vivo assays known in the art.

[0072] In some embodiments, the nucleotide sequence coding for an cc-l,2-mannosidase or a functional fragment is derived from the Trichoderma reesei cc-l,2-mannosidase coding sequence. In specific embodiments, the nucleotide sequence codes for the Trichoderma reesei cc-l,2-mannosidase described by Maras et al. J. Biotechnol. 77: 255-63 (2000), or a functional fragment thereof (such as the C-terminal catalytic domain of the full length protein).

[0073] In most embodiments, the strains are engineered such that the cc-l,2-mannosidase or a functional fragment are targeted to the ER. In specific embodiments, the ER-targeting is achieved by including an ER-targeting sequence in the cc-l,2-mannosidase or a functional fragment. Examples of ER-targeting sequences, i.e., sequences that target a protein to the ER so that the protein is localized or retained in the ER, include an N-terminal fragment of S. cerevisiae SEC12, an N-terminal sequence of S. cerevisiae -glucosidase I encoded by GLSl, and an N-terminal fragment of S. cerevisiae cc-l,2-mannosidase encoded by MNS1. See, also, Nett et al. (2011), supra. In a specific embodiment, the cc-l,2-mannosidase or a functional fragment is targeted to the ER by including an ER-retention signal, HDEL (SEQ ID NO: 91), at the C-terminal of the cc-l,2-mannosidase or its functional fragment.

[0074] The nucleic acid coding for an cc-l,2-mannosidase or a functional fragment can be introduced through an expression vector into a Pichia pastoris strain. The expression vector can be an integrative vector designed to integrate cc-l,2-mannosidase coding sequence into the host genome; or a replicative vector (e.g., a plasmid) which replicates in the strain independent of the chromosomes. In cases of an integrative vector, the vector can be designed to achieve integration of the nucleic acid into the wild type OCHl allele (e.g., through single or double cross over homologous recombination) and simultaneous disruption of the wild type OCHl allele.

[0075] SuperM5 strains

[0076] This disclosure provides Pichia pastoris strains that are robust, stable, and

transformable, and produce proteins with substantially homogeneous Man5GlcNAc2 N- glycans. These strains are also referred to herein as SuperM5 or SuperMan5 strains.

[0077] SuperM5 strains are genetically engineered to include a mutant OCHl allele which is transcribed into an mRNA coding for a mutant OCHlprotein that contains a catalytic domain substantially identical to that of the wild type OCHl protein, but lacks an N-terminal sequence necessary to target the OCHl protein to the Golgi apparatus. The strains do not include any other OCHl allele that produces an mRNA coding for a functional OCHl protein. The strains are additionally genetically engineered to contain a nucleic acid coding for and expressing an cc-l,2-mannosidase or a functional fragment thereof, which is targeted to the ER and converts Man8GlcNAc2 to Man5GlcNAc2.

[0078] An example of a SuperM5 strain is described in Example 1. The nucleotide sequence of the OCHl locus of this strain is set forth in Table 1 and SEQ ID NO: 1. Constructed using the M5-Blast strain described in Jacobs et al. (2009), the SuperM5 strain is superior over M5- Blast in terms of robust growth, stability, and homogeneity of the M5 glycans produced.

[0079] Genetic engineering - introduction of additional enzymes [0080] The strains can be additionally modified to express other, downstream enzymes (or functional fragments thereof) in the glycosylation pathway towards making hybrid- and complex-type N-glycans. Such additional enzymes include, e.g., one or more of GlcNAc transferase I (GnT-I), -l,4-galactosyltransferase 1 (GalT), mannosidase II (Man-II), and GnT-II, among others. See Jacobs et al. (2009); U.S. Patent 7,029,872 to Gerngross.

[0081] GnT-I catalyzes the addition of a -l,2-linked GlcNAc residue to the cc-l,3-mannose of the trimannosyl core in Man5GlcNAc2. Introduction of the GnT-I activity can be achieved by transforming with a vector comprising a nucleic acid sequence coding for a GlcNAc-transferase I (GnT-I) for use in this invention. Such nucleic acid sequence can derive from any species, e.g., rabbit, rat, human, plants, insects, nematodes and protozoa such as Leishmania tarentolae. In specific embodiments, the nucleotide sequence encodes a human GnT-I. The GnT-I or a functional part thereof is targeted to the Golgi apparatus, which can be achieved by including a yeast Golgi localization signal in the GnT-I protein or a functional part thereof. In certain embodiments, the catalytic domain of human GnT-I is fused to the N-terminal domain of S. cerevisiae Kre2p, a glycosyltransferase with a known cis/medial Golgi localization.

[0082] GalT catalyzes the addition of a galactose residue in -l,4-linkage to the β-1,2- GlcNAc, using UDP-Gal as donor substrate. Introduction of the GalT activity can be achieved by transforming with a vector comprising a nucleic acid sequence coding for a GalT or a functional fragment thereof, which can derive from human, plants (e.g. Arabidopsis thaliana), insects (e.g. Drosophila melanogaster). The GalT or a functional part thereof is genetically engineered to contain a Golgi-retention signal and is targeted to the Golgi apparatus. An exemplary Golgi-retention signal is composed of the first 100 amino acids of the Saccharomyces cerevisiae Kre2 protein.

[0083] Man-II acts to remove both terminal a- 1,3- and cc-l,6-mannoses from

GlcNAcMan ₅ GlcNAc ₂ N-glycans. The presence of a terminal -l,2-linked GlcNAc residue on the cc-l,3-arm is essential for this activity. Introduction of the Man-II activity can be achieved by transforming a strain with a nucleic acid vector coding for a Man-II protein or a functional fragment thereof, engineered to contain a Golgi-localization signal. As an example, a suitable nucleic acid can encode the catalytic domain of Drosophila melanogaster Man-II, fused in frame to the Golgi-localization domain of S. cerevisiae Mnn2p. [0084] GnT-II catalyzes the addition of a second -l,2-linked GlcNAc residue to the free cc- 1,6-mannose of the trimannosyl core. Introduction of the GnT-II activity can be achieved by transforming with a vector which contains a nucleotide sequence coding for a GnT-II protein or a functional fragment thereof. GnT-II genes have been cloned from a number of species including mammalian species and can be used in the present invention. As an example, a suitable nucleotide sequence codes for the catalytic domain of rat GnT-II fused to the N- terminal part of S. cerevisiae Mnn2p.

[0085] Other Manipulations to the Strains

[0086] The strains disclose herein can include additional features, achieved by various suitable manipulations (such as cross or recombinant engineering), including, e.g., having a mutant auxotroph gene (e.g., his-) to facilitate cloning and selection, having protease deficiency for limiting product degradation (e.g., pep4-, prbl-, and/or sub2-), having a slow methanol utilization phenotype (e.g., mutS).

[0087] In specific embodiments, this disclosure provides the following strains:

SuperMan5, P. pastoris, ochl-, blasticidin resistant, Mannosidase I from T. reesei (=His+); SuperMan5 (his-), P. pastoris, ochl-, his4-, blasticidin resistant, Mannosidase I from T. reesei;

SuperMan5 (mutS), P. pastoris, ochl-, blasticidin resistant, Mannosidase I from T. reesei (slow methanol utilization);

SuperMan5 (pep4-), P. pastoris, ochl-, blasticidin resistant, Mannosidase I from T. reesei (protease deficient);

SuperMan5 (prbl-), P. pastoris, ochl-, blasticidin resistant, Mannosidase I from T. reesei (protease deficient);

SuperMan5 (pep4-, sub2-), P. pastoris, ochl-, blasticidin resistant, Mannosidase I from T. reesei (protease deficient);

SuperMan5 (pep4-, prbl-), P. pastoris, ochl-, blasticidin resistant, Mannosidase I from T. ressei (protease deficient).

[0088] Use of the Strains

[0089] A heterologous protein with one or more N-glycosylation sites can be expressed in the strains of this invention by transforming a strain of this invention with an expression vector coding for the heterologous protein, to obtain a preparation of the heterologous protein substantially homogeneous in its N-glycan structures.

[0090] EXAMPLE 1 - GENERATION OF A SuperM5 STRAIN

[0091] This Example describes the creation of a SuperMS strain from a M5-Blast strain described in Jacobs et al. (2009), Nature Protocols 4:58-70 (incorporated herein by reference).

[0092] The M5-Blast strain is a modification of the P. pastoris GS115 strain wherein the endogenous mannosyltransferase gene OCH1 is disrupted by the insertion of a vector comprising an a- 1,2 mannosidase gene (pGlycoSwitchM5-Blast vector) through single crossover homologous recombination. As a result of the single crossover homologous recombination, the integrated mannosidase expression cassette is flanked by approximately 450 bp of homologous sequences from the OCHl ORE. The sequence of the OCHl genomic locus of this M5 -Blast strain is set forth in SEQ ID NO: 53. Sequencing revealed the loss of 10 bp at the junction between the pGlycoSwitchM5-Blast vector sequence and the OCHl ORF 3' fragment, resulting in the loss of one of the three desired stop codons from pGl.ycoSwitchM5- Blast vector upstream of the OCHl C -terminal fragment, and frame shifted the second and third stop codons to a different reading frame than the fragment. As a result, the actual ORF was extended 28 bp upstream to an in-frame ATG codon in the vector backbone. Phe27 of the wild type protein became Phel 1 of the new ORF, and the new predicted signal sequence consists partially of the old signal anchor and new, fused sequence from the vector backbone. The amino acid sequence of this new ORF is set forth in SEQ ID NO: 3 (with the first 25 amino acids being the predicted new signal sequence),

[0093] The N-terminal region of the OCHl genomic locus after the single crossover homologous recombination event is diagrammed in Figure 1, along with the construct used to remove this N-terminal region by double crossover homologous recombination. The construct contained both selection and counter-selection markers flanked by a lox71~lox66 pair, allowing for subsequent removal of the selection/counter-selection cassette by ere mediated recombination. The sequence of the double crossover selection/counter-selection cassette with homology arms is set forth in SEQ ID NO: 58, and its creation is described below in this Example, [0094] In order to confirm the sequence of the targeted region prior to creating the crossover construct, PGR primers were designed to amplify -1650 bp of DNA encompassing the region upstream of the mannosidase ORE Using Phusion polymerase (NEB), PGR primers 80670918 and 80670919 amplified an appropriate sized fragment from M5-Blast genomic DNA. The PGR product was TOPO cloned and sequence verified. The DNA sequence demonstrated that the mannosidase expression vector had integrated into the GS1.15 genome correctly at this end of the insert on.

[0095] Flanking PGR primers were designed to amplify the homology regions shown in Figure 1 from M5-Blast genomic DNA. The alignment of these PGR primers is shown in Figure 2. Use of Phusion polymerase resulted in successful PGR reactions from M5-Blast genomic DNA.

[0096] PGR products for the following primer pair combinations were gel isolated and used as templates for the addition of loxJl and lox66 recombination sites:

80765855-80765856 (642 bp, Figure 2A)

80765857-80765858 (658 bp, Figure 2A)

80765852- 80765854 (910bp, Figure 2B)

80765853- 80765854 (956 bp, Figure 2B)

[0097] Mismatch PGR primers were designed to add the lox sites at the appropriate ends of the two homology arms, These mismatch primers are diagrammed in Figure 3. PGR reactions with Phusion polymerase were successful in generating the correct sized DNA products from each of the 3 reactions:

80765855-80984794 (670 bp, Figure 3A)

80765857-80984794 (681 bp, Figure 3 A)

80984795-80765854 (850 bp, Figure 3B)

[0098] In addition to adding lox sites to the arms, PGR primers were designed to add appropriate M5--Blast Pi < hi a genomic DNA extensions onto an existing lox71-MazF~- cassette. Again, Phusion polymerase was used to generate the correct PGR product, as shown in Fi gure 4. The primer pair used: 80984793-80984796 (2941bp, Figure 4)

[0099] The PCR product of the selection/counter- selection cassette was gel purified and a three piece overlap PCR. was performed to attach the homology arms to the cassette.

Briefly, the three pieces were cycled 20x in the absence of primers to anneal and extend the overlap at the ends of the fragments. The cycled mix was then diluted and cycled 35x in the presence of the primers diagrammed in Figure 5.

[00100] The PCR reaction was performed with Phusion polymerase, using an extension time of 3 min. Primers are detailed below:

80765855-80765854 (4311 bp, Figure 5)

[00101] This PCR product was gel isolated and TOPO cloned. Selection of the TQPQ clonmg was performed on LB-Nat plates to ensure the inclusion of the selection cassette. DNA sequencing was performed on multiple isolates to determine the homology arm sequences. The final isolate contained a functional Nat ^R expression cassette, the lox71 and lox66 recombination sites and the correct homology arms.

[00102] PCR primers internal to the cloned fragment detailed in Figure 5 were used to generate linear DNA for Pichia pastoris transformation. Two independent sets of primers were designed:

81364233-81364234 (4063 bp, Figure 6)

81364235-81364236 (4060 bp, Figure 6)

[00103] PCR reactions were performed using Phusion polymerase with an extension time of 100 sec.

[00104] The PCR products were purified by agarose gel electrophoresis and eluted from the binding matrix with water. The M5-Blast Pichia pastoris strain was made competent for electroporation using a standard DTT / sorbitol treatment. Electroporation was performed using 1 mm cuvettes containing 20 μΐ competent cells and 1-2 μΐ of purified linear DNA. Transformation mixtures were plated on YPD-Nat agar.

[00105] After electroporation, cells were grown out at 30°C for 3 days. Individual transformants were patched to YPD-Nat for storage and analysis. Figure 7 shows the theoretical arrangement of the OCHl locus after proper double crossover integration of the PGR produet(s) into the M5-Blast genome. PGR primer pairs were designed to check that the nourseothricin-resistant isolates were the result of homologous recombination, rather than random integration of the PGR. product(s) into the genome. These PGR. primer pairs are diagrammed on Figure 7.

81487116-81487117 (895 bp. Figure 7)

81487118-81487119 (937 bp. Figure 7)

81487120-81487121 (656 bp, Figure 7)

81487122-81487123 (756 bp, Figure 7)

[00106] A total of 24 independent isolates were screened by PGR and 2 isolates that appeared correct were further characterized by DNA sequencing of the PGR products. The two isolates were struck to single colonies on YPD medium and retested on YPD-Nat. Small scale genomic DNA preparations were made using phenol/chloroform glass bead lysis. Based on the sequencing results of the 81487116-81487117, 81487118-81487119, 81487120-81487121 and 81487122-81487123 primer pairs on these genomic extracts, both isolates contained the lox71-lox66 selection/counter-selection cassette at the proper location in the M5--Blast genome. There were no mutations introduced by the initial PGR reaction to generate the transformation fragment, the recombination junctions at both ends were identical to M5-Blast "wild- type" DNA sequence, and both the lox.71 and lox66 sites were intact. The DNA sequence of the OCHl locus after double cross over recombination is set forth in SEQ ID NO: 59.

[00107] The two isolates (A 1-2 and A4-3) were transformed with a plasmid constitutively expressing ere recombinase. Briefly, both strains were made electro- competent using a DTT/ sorbitol procedure, electroporated with circular plasmid and plated on YPD-G418. Transformants were grown out at 30°C for several days and colonies picked. Colonies were either transferred directly to methanol plates to induce the MazF counter-selection or patched to YPD to allow loss of the cre-ARS plasmid prior to MazF' induction. Methanol induction was carried out on both BMMY (1% methanol) and GSM (complete synthetic medium, 0.5% methanol). Plates were supplemented with methanol daily by adding 100 μΐ methanol to the inverted plate lid. Incubation was carried out at 30°C. There was significant colony formation under all conditions tested; growth on methanol appeared independent of whether the transformant came directly from YPD- G418 or had undergone an intermediate patching on YPD without G418.

[00108] Cre recombination should remove the DNA sequences between the lox71 and lox66 sites, leaving only a defective lox site scar in the genome. The theoretical result of this recombination event is shown in Figure 8, PCR primers were designed to amplify the region containing the defective lox scar. Twenty colonies that grew on methanol were screened by PCR to determine the loss of the selection/counter--- selection cassette. PCR primers used were:

80670916-80670917 (680 bp, Figure 8)

80670918-80670919 (782 bp, Figure 8)

[00109] Seventeen of twenty isolates generated the appropriate PCR product with the first primer pair. Most, but not all, of the 17 also showed an appropriate product with the second primer pair. Each of the 17 isol ates was patched to YPD, YPD -Blast, Y PD-Nat and YPD-G418 to test for the presence or absence of the drug selection markers. If the cre plasraid had properly removed the selection/counter-selection cassette and subsequently been lost, the resulting strain should be blasticidin resistant and sensitive to both G418 and nourseothricin. All isolates were blasticidin resistant and nourseothricin sensitive. A few retained G418 resistance (still contained the cre plasmid, perhaps integrated) and were discarded. Of the remainder, 4 were picked for DNA sequencing of the LEU5- mannosidaseHDEL intereenic region.

[00110] Existing PCR primers were used to amplify the genomic region spanning

LEU 5 and the mannosidaseHDEL ORE

81487118-80765854 (1602 bp, Figure 9)

[00111] PCR amplification was performed using Phusion polymerase on genomic

DNA that had been prepared by phenol/chloroform glass bead extraction. Multiple internal sequencing primers were used to verify the entire sequence of the 1602 bp PCR product. All 4 of the sequenced PCR products were correct, and contained a defective lox site at the proper location between the LEU 5 gene and the mannosidaseHDEL ORF. Both the LEU 5 promoter and the GAP promoter driving mannosidaseHDEL expression were intact and identical to the promoters present in the starting M5--Blast strain. The DNA sequence of the OCH1 locus after double crossover recombination and ere recombination is set forth in SEQ ID NO: 1.

[00112] Glycerol stocks of each of the 4 isolates (and 2 parental strains prior to ere recombination) were prepared , bG yeast-100015 A 1-2 (pre-recombinatiom)

bG yeast-100016 A4- 3 (pre-recombination)

bG yeast-100017 isolate 1 (post-recombination)

bG yeast- 100018 isolate 2 (post-recombination)

bG yeast-100019 isolate 3 (post-recombination)

bG veast- 100020 isolate 4 (post-recombination)

[00113] Each glycerol stock was streaked and retested for the appropriate markers: bG yeast-100015 his ^" , blasticidin ^R , nourseothricin ¹ *

bG yeast-100016 his ^" , blasticidin ^R , nourseothricin ^R

bG yeast ! 0001 7 his ^" , blasticidin ⁸ -, nourseothricin ⁸

bG yeast- 100018 his ^" , blasticidin ^R , nourseothricin ⁸

bG yeast-100019 his ^" , hlasticidin ^R , nourseothricin ⁸

bG veast-100020 his ^" , blasticidin ^R , nourseothricin ⁸

[00114] All glycerol stocks tested as expected.

[00115] YPD stabs of all 6 isolates were generated and subjected to glycoanalysis.

Glycerol stock bG yeast- 100017 was used to generate a large genomic DNA preparation for genomic sequencing. In addition, samples were prepared from wild-type GS115 and the M5-Bl.ast strain. Briefly, cell pellets from 100 ml yeast cultures (YPD, 30°C growth) were resuspended in 1 M sorbitol/100 niM citrate (pH 6.0) and treated with Zymolyase

(Zymo Research) containing RNase for 2 h at 37°C. SDS was added to 0.5% to lyse spheroplasts. Proteinase K was then added and the mixture incubated at 50°C overnight.

An equal volume of phenol/chloroform was added and the mixture gently rocked for 30 min. After centrifugation, the upper aqueous layer was removed and DN A precipitated with isopropanol, The threaded DNA was spooled from the solution and resuspended in

TE. The DN A was reprecipitated with ethanol. and then washed with 70% ethanol, air- dried and resuspended a final time in TE.

[00116] DNA was distributed in multiple tubes: bG DNA- -100215 GS115 genomic DNA

bG DNA- -100216 GS115 genomic DNA

bG DNA- -100217 GS115 genomic DNA

bG DNA- -100221 bG yeas-100017 genomic DN A

bG DN A -100222 bG yeas t-100017 genomic DNA

bG DNA- -100223 M5-Blast genomic DNA

bG DNA- -100224 M5-Blast genomic DN A

[00117] In order to test the genomic DNA isolates and verify that the manipulations performed in creating the bG yeas -100017 strain had not altered the mutant form of the OCHl ORF, the N-terminal region of the OCH! ORF was isolated from bG DNA-100221 (new strain) and bG DNA- 100223 (M5- Blast strain) by PGR and resequenced, Both DNA preparations were identical at the OCHl ORF locus, and contained the 10 bp deletion as described above.

[00118] Primers used in this Example are listed below:

SEQ ID

60 30670916 CAAGTTGCGCCCCCTGGCA

61 80670917 TGGAGCAGCTAATGCGGAGGA

62 80670918 AGTTCCGCCGAGACTTCCCCA

63 30670919 1 T C A G C C G G A AT TTGTGCCGT

64 80765852 ATCCAGGGTGACGGTGCCGA

65 80765853 GCAAGAGGCCCGGCAGTACC

66 30765854 CCGCCCTCGT A G G G T T G G G A G

67 80765855 TTCGCGGTCGGGTCACACA

68 80765856 AACTGCCATCTGCCTTCGCC

69 30765857 C AAA TCGCGGGTTCGCGGTC

70 80765858 GAGCAAACTGCCATCTGCCTTCG

71 80984793 GTGTTCGTAGCAAATATCATCAGCCTACCGTTCGTATAGCATACATTA

T AC G A A G T T A . T G G A T C T A AC A T C C A A A

72 80984794 TTTGGATGTTAGATCCATAACTTCGTATAATGTATGCTATACGAACGG

T AGG C T GAT GAT AT T T GC T AC G A_AC AC 73 80984795 GCCGCCATCCAGTGTCATAACTTCGTATAGCATACATTATACGAACGG

T AC T T T T T T G T A G A A A T G T C T T G G T G T

74 80984796 ACACCAAGACATTTCTACAAAAAAGTACCGTTCGTATAATGTATGCTA

TACGAAGT TATGACACTGGATGGCGGC

75 81364231 GT G T T C GT AG C AAAT AT C AT CAGC C T AC C G

76 81364232 AC AC C A AG AC A T T C TAG A A A A A A G T A.C C GT

77 81364233 T T CGCG GT CGGG T C AC AC AC

78 81364234 GGAGCAGC TAAT GC GGAGG ATGC

79 81364235 CGGTCGGGTCACACACGGAG

80 81364236 T G G A G C A G C T A A T G C G G A G G A

81 81487116 T GAG TCCTGGTGCTCCT G AC G

82 81487117 CCCCTCCTGTTGCGTTTGGC

83 81 87118 AGCGTT C T GAGT C C TGGTGC T

84 81487119 GGTC C T GC GT T T GC AAC GG T

85 81487120 A C T A AC G C C G C C A T C C AG T G T C

86 81487121 GC T T CAGC C GGA AT T T GTGCCG

87 81487122 C GC C T C GAG AT CAT C T GC C C

88 81487123 T CAGCC GGAAT T T GT GC C GT

EXAMPLE 2 - STORAGE AND HANDLING

[00119] SuperM5 was stored in different conditions at -80°C, -4°C, 20°C and at room temperature. Strains were stored as frozen glycerol stocks and as stab cultures. Different cultures were stored and thawed for different experiments and for shipping to collaborators for testing. In all cases the strains recovered, plated and cultured similar to the parent Pichia pastoris GS115 strain and grew in both complex and defined media similar to the parent strains. The SuperM5 strains transformed similarly as the parent strain and proteins were expressed with the mannose-5 glycosylation as the predominate glycoform, or the only glycoform. Strains have been repeatedly stored and regrown to establish robustness of the SuperM5 strains.

EXAMPLE 3 - ANALYSIS OF TEST PROTEINS IN P. PASTORIS STRAINS

[00120] The genes for Candida antartica lipases A and B, human transferrin, and the human CH2 domain from IgG were integrated into the SuperM5 genome using standard

transformation methods. In all cases significant amounts of protein were produced and secreted into the medium. Transformed strains and media-containing protein were tested for glycan analysis using previously published methods. In all cases, the glycan profiles for the test proteins and for the strain glycoproteins demonstrated a mannose-5 glycan structure with no other higher mannose structures detected by the methods used.

EXAMPLE 4 - ANALYSIS OF CELL WALL MANNOPROTEINS IN P. PASTORIS STRAINS

[00121] Twelve Pichia pastoris strains and the Man5-Blast strain were started in a 24- well plate containing 2ml YPD and grown overnight at 28 °C while shaking (250 rpm). After growth, cells were harvested by centrifugation (3000g for 5 min at room temperature) and cell wall mannoproteins were extracted according to the protocol by Jacobs et al. (see Jacobs et al., 2009, Nature Protocols 4(l):58-70). The extracted mannoproteins (in 100 μΐ ddH20) were diluted to 300 μΐ with RCM buffer (8 M urea, 3.2 mM EDTA, 360 mM Tris-HCL, PH 8.6). N-glycans were prepared from these samples following the 96- well on-membrane deglycosylation procedure as published by Laroy et al. (Laroy et al., 2006, Nature Protocols, 1: 397-405).

[00122] After labeling the dried N-glycans with 8-aminopyrene-l,3,6-trisulphonic acid2, the excess of label was removed using size exclusion chromatography (Sephadex G-10 resin2). The samples were finally reconstituted in 10 μΐ of ultrapure water and diluted lOx prior to their injection (80" at 1.2 kV) in the capillaries (e.l. 36 cm; i.d. 50 μιη) of an ABI 3130 DNA sequencer. The following settings were applied: Oven temperature: 60°C Run voltage: 15 kV; Prerun voltage: 180" Run time: 1000"; Prerun time: 15 kV. The Genemapper v3.7 was used to analyze the obtained data and structures were assigned to the peaks (see Figure 10).

EXAMPLE 5 - MATERIALS AND METHODS

[00123] Below describes non-limiting examples of materials and methods for the present invention.

[00124] Plasmids and strains: Pichia pastoris expression vector pPICZaA was purchased from Invitrogen Corporation; pUC19/GM-CSF plasmid (containing GM-CSF Gene sequence) was synthesized by Shanghai Qing-Lan Biotech Co., Ltd.; Saccharomyces cerevisia expression vector pYES2, Pichia pastoris X-33 (wild Type), E. coli JM109 were from the inventors' laboratory.

[00125] Reagents and instruments: Taq DNA polymerase, Pfu DNA polymerase, restriction enzymes, T4 ligase, 5 - fluoroorotic acid (5-FOA) was purchased from Shanghai Biological Engineering Technology Services Co., Ltd.; Zymolyase was purchased from Sigmag company (USA); N-glycosidase F (PNGase F) was purchased from New England Biolabs, Inc. (USA); peptone, yeast extract, yeast nitrogen base without amino acids (YNB) were purchased from BIO BASIC INC (Canada). PCR machine (PTC 100) was from MJ Research, Inc. (USA); electrophoresis systems, gel imaging system were from Bio-Rad (USA); AKTA purification system purchased from GE (USA).

[00126] Primers: based on the reported Pichia URA3 (orotidine-5 '-Phosphate decarboxylase) gene sequence (GenBank: AF321098), two pairs of extension amplification primers based on homologous fragment were designed: URA5F, URA5R and URA3F, URA3R; based on Saccharomyces cerevisiae expression vector pYES2 sequence, primers pYES2F and pYES2R were designed; based on the GenBank (E12456) reported Pichia OCH1 gene sequence, two pairs of amplification primers based homologous sequence were designed: OCH5F, OCH5R and OCH3F, OCH3R. The internal identification primers (in) 5F, (in) 3R were also based on the same sequence; universal primers 5' AOX1, 3' AOX1 sequences were based on references. Primers were synthesized by Shanghai Biological Engineering Technology Services Co., Ltd.

[00127] Yeast cell culture, genomic extraction and PCR conditions were performed based on known protocols.

[00128] The construction of URA3 homologous replacement DNA sequence: using the X-33 strain genome as a template and primer pairs URA5F, URA5R and URA3F, URA3R, the homologous fragments of both sides of URA3 genes, URA5' and URA3', a 700 bp and a 600 bp, respectively, were PCR amplified. Then using URA5' and URA3' as templates and URA5F and URA3R as a primer pair, the URA5-3, the target homologous replacement DNA fragment for URA3 gene was PCR amplified, which was about 1300 bp in size. [00129] The construction of pYXZ plasmid: using plasmid pYES2 as a template and primer pair pYES2F and pYES2R, the sequence that contains URA3 gene was PCR amplified. The PCR product was purified and digested with Sal I and followed with ligation reaction. The self-ligased plasmid pYXZ was transformed into E. coli JM109, and plated on LB plates containing ampicillin to select positive clones.

[00130] The cloning of OCH1 homologous arm: using the X-33 strain genome as a template and primer pairs OCH5F, OCH5R and OCH3F, OCH3R, to PCR amplify the 5' and 3' ends of the OCH1 gene homologous arms, OCH5' and OCHJ'and its fusion fragment OCH3-5. The method used was similar to what has been described above. The fragment sizes were 1000 bp, 700 bp and 1700 bp, respectively.

[00131] The construction of Knockout plasmid pYXZ-OCHl: the inventors digested the OCH1 gene 5' and 3' homologous fusion fragment OCH3-5 with Nhe I and Sal I and cloned the fragment into pYXZ plasmid digested with Sal I and Nhe I to make the knockout plasmid pYXZ-OCHl.

[00132] Knockout the URA3 gene from Pichia pastoris X-33 to construct auxotrophic selection marker: X-33 competent cells were shock transformed using the fusion fragment URA5-3 arm that has homologous sequence to both ends of the URA3 gene; the transformed cells were spread on MD medium containing 5-FOA and uracil (YNB 1. 34%, glucose 2%, agar 1.5%, uracil 100 μg / mL, 5-FOA 1 mg / mL), and incubated at 30 degrees Celsius for 3 - 5 days. Single colonies grown on the medium were selected and seeded with a toothpick, respectively, to MD medium (YNB 1. 34%, glucose 2%, agar 1.5%) and MDU medium (YNB 1. 34%, glucose 2 %, agar 1.5%, uracil 100 μg / mL), and incubated at 30 degrees Celsius for 3 - 5 days. Then, strains that grew well on the MDU medium but could not grow on the MD medium were selected. The selection process was repeated for 3 rounds to get stable traits and the final strains were confirmed by PCR reaction using URA5F, URA3R as primers and genomic DNA as template.

[00133] OCH1 gene knockout of Pichia pastoris X-33: the knockout plasmid pYXZ-OCHl was linearized at Mlu I site that is located between the two homologous arms and electric shock transformed into the X-33 (ura3-) competent cells, and spread on MD medium, and incubated at 25 degrees Celsius for about a week. Single colonies were picked with a toothpick and seeded to the same coordination on two plates with YPD medium (peptone 2%, yeast extract 1%, glucose 2%, agar 1.5%), and incubated at 25 degrees Celsius and 37 degrees Celsius, respectively for a few days. The colonies that grew well at 25 degrees Celsius but could not grow at 37 degrees Celsius were extracted to obtain genomic DNA. OCH1 gene external primers OCH5F, OCH3R and internal primers (in) 5F, (in) 3R were used for PCR identification.

[00134] Construction of expression vector: the plasmid pUC19/GM-CSF from the inventors' own laboratory was double digested with EcoRI and Not I. The GM-CSF gene fragment was extracted (a 6xHis tag sequence was introduced), and cloned into Pichia pas tons expression vector pPICZaA digested with the same restriction enzymes to make the expression vector pPICZaA/GM-CSF. Positive clones were selected and confirmed with restriction enzyme digestion and sequencing.

[00135] The expression and analysis of GM-CSF in Pichia pastoris X-33 and X-33 (ochl-): linearize the expression vector pPICZaA/GM-CSF with Sal I and electrically shock transformed the plasmid into X-33 and X-33 (ochl-) competent cells. Shock mixture was spread to culture cloth coated with YPDZ medium (each containing 100 μg/mL, 300 μg/mL, or 500 μg/mL Zeocin), the X-33 transformants were grown at 30 degrees Celsius for 3-5 days, and X-33 (ochl-) transformants were cultured at 25 degrees Celsius for about a week. Single colonies that grew well were picked to extract genomic DNA and identified with PCR reaction using primers 5ΆΟΧ, 3ΆΟΧ1 to select positive transformants. Positive X- 33/PICZaA/GM-CSF cells were inoculated into 2 mL of YPD medium (2% peptone, 1% yeast extract, 2% glucose), incubated at 30 degrees Celsius for 24 h. The culture was used to inoculate (5% inoculation ratio) into 10 mL of BMGY medium (2% peptone, yeast extract 1%, YNB 1. 34%, glycerol 2%, 100 mmol / L phosphate buffer, pH 6.0). After incubation at 30 degrees Celsius for 36 h, the culture was centrifuged to remove the supernatant and the pellet was resuspended to 3 mL of BMMY medium (yeast extract 1%, YNB 1.34%, peptone 2%, 100 mmol / L phosphate buffer, PH 6. 0), 2% methanol was added to induced

expression: X-33 (ochl-)/pPICZaA/GM-CSF positive cells were cultured in the YPD medium at 25 degrees Celsius for 48 h, BMGY at 25 degrees Celsius for 48 h, and induced expression at 25 degrees Celsius. Expression induction condition was same as that used in X- 33 cells, methanol was added every 24 h and the induction was for 72 h. Once it was finished, the cell cultures were centrifuged and supernatant was collected for protein analysis.

[00136] EXAMPLE 6 - TRANSCRIPTOME ANALYSIS OF M5-BLAST AND

SUPERM5 STRAINS

[00137] Strain Growth For RNA Isolation. BG10, GS115, M5 Blast and SuperMS (described in Example 1) strains were maintained on YPD Agar plates as patches. For transcriptome analysis, a 50 ml culture of each strain was inoculated from a patch and grown in BMGY at 30°C, 200 rpm for approximately 16 hours. The stationary culture was diluted 100-fold into fresh BMGY medium and grown at 30°C, 200 rpm for 6 hours. This time point was considered exponential growth with glycerol as the carbon source. Aliquots were spun down in 15 ml tubes, supernatants discarded and the cell pellets rapidly frozen in liquid nitrogen. Cell pellets were stored at -80°C for subsequent total RNA isolation.

[00138] Total RNA Isolation. FastRNA SPIN kits (MP Bio) were used to isolate total RNA. Cell lysis was per-formed using a BioSpec Mini-Beadbeater 96. Total RNA was eluted from the spin column in 15 μΐ of RNase/DNase-free water, frozen in liquid nitrogen and stored at -80°C. RNA samples were shipped on dry ice for RNA-Seq analysis on an Illumina HiSeq machine. RNA samples were analyzed using an Agilent Bio Analyzer, and all showed intact yeast ribosomal RNA peaks.

[00139] RNA Library Generation and Sequencing. mRNA libraries were prepared using Illumina reagents. A TruSeq RNA Sample Preparation Kit was used to selectively generate bar-coded cDNA from poly A RNA. After bar-coding and amplification, a total of 12 samples were pooled (4 samples for this study) for analysis. Fifty base, single end reads were performed. Data was supplied to BioGrammatics in standard FASTQ format. Reads were trimmed based on ambiguous bases and quality score and then filtered to eliminate all trimmed reads that were less than 40 bases in length. Approximately 0.3% of reads were removed from each data set.

[00140] The RNA-Seq algorithm of CLC Genomics Workbench was used to map the reads from each data set to the BG10 wild type annotated sequence. Note that the BG10 genome does not contain the expression cassettes for the mannosidase and biasticidin resistance gene present in the Man5 and SuperMS strains. [00141] Gene Expression Profiling. Expression profiles from each of the 4 strains were plotted and clustered. Scatter plots (with R-values) were evaluated for strain to strain comparisons of overall expression profiles, 'The BG10 and GS115 strains show the tightest correlation (R-value - 0,98), followed by the Man5 and SuperMS strains (R-value = 0.95). A slight general upregulation was observed in the OCH1 mutant strains vs. GS115 (R-values of 0.92 and 0.84 for Man5 and SuperMS respectively). Overall, gene expression patterns are similar amongst the 3 strains (GS115, M5 and SuperMS) when grown on glycerol.

[00142] From each of the RNA-Seq data sets mapping to the BG10 strain, the OCHl mapping was extracted. In the BG10 and GS115 strains, the coverage scale was from 0 to about 75. The expression levels of OCHl were approximately equal. Sequencing reads were distributed approximately equally across the open reading frame, The expression levels of OCHl in these two strains were approximately 0.2% that of the most highly expressed genes,

[00143] For the SuperMS strain, the coverage scale was from 0 - 47. The expression level dropped to approximately half that of the BG10 and GS115 strains. Also, there was no coverage of the N-terminus of the open reading frame. This lack of coverage was the result of the complete deletion of these DNA sequences from the SuperMS strain.

[00144] For the ManS strain, the coverage scale was from 0 - 502, There was significantly more coverage of the N-terminal portion of the open reading frame than the C-terminal portion. This disjointed coverage was the result of the duplication of most of the N-terminal portion of the open reading frame in the ManS strain. The N-terminal portion of the ORF is expressed from DNA upstream of the mannosidase ORF and the mutant form of the C- terminal portion of the ORF was expressed downstream of the mannosidase and blasticidin resistance ORFs. Based on read coverage, the C-terminal portion of the ORF appears to be slightly less abundant in ManS than in SuperMS,

[00145] Mapping of the ManS and SuperMS data to the mutant form of the OCHl ORF shows complete coverage of the mutant OCHl ORF in both strains, indicating gene expression, The Man strain shows extra coverage in the N-terminal portion of the ORF ^' , for the same reasons described above for the wild type OCHl ORF mapping,

[00146] Mapping of the ManS and SuperMS data to the mannosidase ORF shows similar expression levels in the two strains.

[00147] Conclusion. Transcriptome analysis has been performed on the GS115, Man and

SuperMS strains. The strains show similar overall gene expression patterns. In the Man and SuperMS strains, a mutant form of the OCHl ORF is expressed (polyadenylated mRNA is present). The mannosidaseHDEL ORF is expressed in both strains at approximately the same level.

[00148] EXAMPLE 7 - Trastuzumab Expression in a M5-Blast like and SuperM5 Strains

[00149] In Study 1, the SuperM5 strain described in Example 1 was transformed with an expression vector coding for trastuzumab by electroporation. Zeocin-resistant colonies were screened by the genome PCR using AOX1 primers and Herceptin specific primers. Positive clones of the genome PCR was cultivated and Man5-type trastuzumab expression to the culture supernatants was evaluated by SDS-PAGE.

[00150] In Study 2, Pichia strains transformed with an expression vector coding for trastuzumab were screened to select a strain that expressed high levels of trastuzumab. The selected strain was transformed with GlycoSwitch® plasmid (pGlycoSwitch-M5/2 (GAP, BSD), provided by Gent University) by eletroporation. Blasticidin S-resistant colonies were screened by the genome PCR for detecting the pGlycoSwitch-M5 insertion into the OCHl locus and MDS1 gene presence. Positive clones of the genome PCR was cultivated and Man5-type trastuzumab expression to the culture supernatants was evaluated by SDS-PAGE.

[00151] In Study 3, the positive clones obtained in Study 1 (clone 46) and Study 2 (clone 11) were cultivated in a 1L baffled flask. Trastuzumab expression was induced by replacing with methanol containing medium. 72 hours after methanol induction, trastuzumab was purified using Protein A-affinity resin from the culture supernatants. Productivity of trastuzumab from clone 46 and clone 11 was 3 mg/L and 1.3 mg/L culture, respectively.

[00152] In Study 4, the N-glycan structures of trastuzumab produced in clone 46 (Study 1) and clone 11 (Study 2) were analyzed. The homogeneity of N-glycan structures was assessed in the primary analysis, and the N-glycan structures were identified in the secondary analysis according to searching N-glycan database and HPLC injection along with the standard sample. From these analyses, the N-glycans of trastuzumab obtained from clone 46 (Study 1) were virtually homogeneous and the predominant (or essentially the only) N-glycan was estimated as Man5GlcNac2 from MALDI-TOF mass analysis (Figure 11). The N-glycan structures of trastuzumab obtained from clone 11 (Study 2) were found to be a mixture of Man5GlcNAc2 to Man8GlcNAc2.

Table 7. N-glycan analysis of trastuzumab obtained from Study 2

[00153] Her2 binding affinity of Man5-type trastuzumab obtained from clone 46 was analyzed in parallel with commercial Herceptin by ELISA and BIAcore assays, was found to have similar HER2-binding activity to the commercial Herceptin. See Figure 12 and Table 8.

Table 8. Kinetic parameters of trastuzumab analyzed on BIAcore mAb k _a (M - ¹ s ^"1 } k _d is ^-1 } _A (NT ¹ ) K _D <nM)

Man5-trastuzumab 2.29 x 10 ⁵ 2.43 x 10 ^"5 1.20 x 10 ¹⁰ 0.083 mAb k _a (NT ¹ s- ¹ ) k _d is ^'1 ) _A CM ^'1 ) K _D (nM)

CHO Herceptin 4.25 x 10 ⁵ 5.21 x 10 ^"5 8.17 x 10 ⁹ 0.12

Pichia trastuzumab 4.65 x 10 ⁵ 8.72 x 10 ^"5 5.33 x 10 ^s 0.19

[00154] EXAMPLE 8 - Analysis of Additional Glycosylated Proteins Expressed in M5- Blast and SuperM5

[00155] Genes for Candida antarctica lipases A and B (CalA, 2 N-glycosylation motifs and CalB, 1 N-glycosylation motif) as well as for human serum transferrin (2 N-glycosylation motifs), driven by an AOXl promoter, were integrated into the genome of the M5-Blast strain as well as the SuperM5 strain, both described in Example 1, via homologous recombination at the AOXl locus (selection by Zeocin). A plasmid harboring a

complementation cassette for histidine auxotrophy next to a synthetic gene coding for native Pichia PDI that is driven by an AOXl promoter, was co-transformed. Selection was done on solid minimal media with Zeocin.

[00156] 47 transformants of each combination described above were cultivated and screened for protein abundance and quality with respect to obvious changes in the migration behavior of the secreted proteins on microCE (capillary electrophoresis, GXII, CaliperLS). Mock strain supernatant (GS115) was applied as negative control.

[00157] All 3 proteins secreted from the SuperM5 strain showed comparable expression levels as compared to the M5-Blast strain. Furthermore, target protein signals from the SuperM5 supernatants on microCE exhibited a lowered migration time as those from M5- Blast supernatants, shifting to lower apparent molecular weights. It is believed that altered N-glycosylation of secreted proteins from SuperM5 resulted in a lower molecular mass in microscale.

[00158] Samples of the supernatants from microscale cultures and those from cultures in a bioreactor were analyzed for its N-glycan compositions. From the samples obtained from microscale culture, 0.5 ml of the medium was diluted with two times the volume of RCM buffer (8 M urea, 3.2 mM EDTA, 360 mM Tris-HCL, PH 8.6). From the bioreactor samples, 0.2 ml medium was used. N-glycans were prepared from these samples following the 96-well on-membrane deglycosylation procedure as published by Laroy et al. (supra). After labeling the dried N-glycans with 8-aminopyrene-l,3,6-trisulphonic acid, the excess of label was removed using size exclusion chromatography (Sephadex G-10 resin). The samples were finally reconstituted in 10 μΐ of ultrapure water and diluted lOx prior to their injection (80" at 1.2 kV) in the capillaries (e.l. 36 cm; i.d. 50 μιη) of an ABI 3730 DNA sequencer. The following settings were applied:

Oven temperature: 60°C Run voltage: 15 kV

Prerun voltage: 180" Run time: 1000

Prerun time: 15 kV [00159] The Genemapper v3.7 was used to analyze the obtained data and structures were assigned to the peaks.

[00160] As shown in Figures 13-15, the N-glycans of proteins produced from the SuperM5 strain were substantially homogeneous, with Man5GlcNAc2 being the principal N-glycan. In contrast, the N-glycans of proteins produced from the M5-Blast were quite heterogeneous, especially from cultures in a bioreactor.

[00161] EXAMPLE 9.

[00162] In this Example, a diploid strain is created by mating the Super 5 strain descri bed in Example 1 and a wild-type Pichia pastoris strain of a different genetic background. The combination of the two genetic backgrounds allows a determination whether second site repressors or enhancers of the OCH1 disruption phenotype exist in either strain. The diploid is "held together" using two dominant selectable markers in identical genomic locations in each haploid strain. At the diploid OCH1 locus this strain transcribes two different mRNAs; one encoding the wild-type Ochlp (from the wild-type haploid genomic copy) and the other encoding the mutant Ochlp (from the Super anS haploid genomic copy).

[00163] A double-crossover vector containing a Hygromycin B selection marker is constructed that replaces a highly conserved region of Ochlp with a V5 epitope tag. This vector is designed so that integration into the diploid genome will, at approximately 50/50 distribution, replace the highly conserved domain in either the wild-type or mutant form of Ochlp, creating both epitope insertions in the same starting genetic background. In the case where the vector integrates into the SuperMS genomic copy of OCHL the drug selection marker on the vector will be tightly linked to the existing Blasticidin marker adjacent to OCHL Genomic PGR and DNA sequencing can be used to verify the construction of the two diploid strains, one with the wild-type and one with the mutant form of Ochlp epitope tagged.

[00164] The diploids are sporulated and random spores grown and analyzed. After growth on non- selective medium, resulting haploid colonies are scored for Hygromycin B resistance. Distribution and growth characteristics of Hygromycin B resistance haploids can determine the lethality or growth deficiency of Ochlp inactivation by epitope insertion.

[00165] Methods - An existing SuperManS strain with a Zeocin resistance marker at the prblA locus is mated with a BG10 haploid strain with a nourseothricin resistance marker at the prhlA locus to create the starting diploid strain. The BG10 strain is created from a prblA knockout DNA construct.

[00166] A Hygromycin B vector is constructed to replace 14 amino acids in the Ochlp sequence ( LF A RGGLY ADMDT ^' M L, SEQ ID NO: 92) with the 14 amino acid V5 epitope (GKPIPNPLLGLDST, SEQ ID NO: 93). This retains the full length coding region for both the wild-type and mutant forms of Ochlp when integrated into the genome,

[00167] The Hygromycin B vector is integrated by homologous recombination into the diploid genome. PGR screening of genomic DNA can be used to verify the chromosome (either SuperManS or BGIO) and location at which homologous recombination has occurred. PGR products from positive strains are sequenced to verify the replacement of the 14 amino acid domain from Ochlp with the V5 tag, making sure the respective ORF lengths are retained in each of the two copies.

[00168] The two strains are grown and spomlated, and resultant haploids verified by sensitivity to one or the other drug marker at the prblA locus. Haploids can be visually screened for growth phenotype at the plate level and, if a marked growth distribution is observed, scored for the presence of the V5 tagged construct at either or both of the

SuperManS or BG10 ochl loci. If all haploids grow equally well, they can be scored for the presence of the Hygromycin B marker. Loss of the Hygromycin B marker on sporulation and subsequent germination will indicate that insertion of the V5 epitope into the Ochlp protein is lethal in both the wild-type and mutant cases.

[00169] Detection of V5 tagged protein by Western blot in supematants and extracts of both diploid strains, and, if viable, resultant haploids. As additional experimentation, subcellular location of the wild-type and mutant V5 tagged forms of Ochlp can be performed by immunofluorescence on diploid cells.

[00170] As used herein, the term "about" refers to plus or minus 10% of the referenced number.

[00171] Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application is incorporated herein by reference in its entirety.

[00172] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims.

[00173] The reference numbers recited in the below claims are solely for ease of examination of this patent application, and are exemplary, and are not intended in any way to limit the scope of the claims to the particular features having the corresponding reference numbers in the drawings.

Table 1. SEQ ID NO: 1.

1 AACGTCAAAG ACAGCAATGG AGTCAATATT GATAACACCA CTGGCAGAGC GGTTCGTACG

61 TCGTTTTGGA GCCGATATGA GGCTCAGCGT GCTAACAGCA CGATTGACAA GAAGACTCTC

121 GAGTGACAGT AGGTTGAGTA AAGTATTCGC TTAGATTCCC AACCTTCGTT TTATTCTTTC

181 GTAGACAAAG AAGCTGCATG CGAACATAGG GACAACTTTT ATAAATCCAA TTGTCAAACC

241 AACGTAAAAC CCTCTGGCAC CATTTTCAAC ATATATTTGT GAAGCAGTAC GCAATATCGA

301 TAAATACTCA CCGTTGTTTG TAACAGCCCC AACTTGCATA CGCCTTCTAA TGACCTCAAA

361 TGGATAAGCC GCAGCTTGTG CTAACATACC AGCAGCACCG CCCGCGGTCA GCTGCGCCCA

421 CACATATAAA GGCAATCTAC GATCATGGGA GGAATTAGTT TTGACCGTCA GGTCTTCAAG

481 AGTTTTGAAC TCTTCTTCTT GAACTGTGTA ACCTTTTAAA TGACGGGATC TAAATACGTC

541 ATGGATGAGA TCATGTGTGT AAAAACTGAC TCCAGCATAT GGAATCATTC CAAAGATTGT

601 AGGAGCGAAC CCACGATAAA AGTTTCCCAA CCTTGCCAAA GTGTCTAATG CTGTGACTTG

661 AAATCTGGGT TCCTCGTTGA AGACCCTGCG TACTATGCCC AAAAACTTTC CTCCACGAGC

721 CCTATTAACT TCTCTATGAG TTTCAAATGC CAAACGGACA CGGATTAGGT CCAATGGGTA

781 AGTGAAAAAC ACAGAGCAAA CCCCAGCTAA TGAGCCGGCC AGTAACCGTC TTGGAGCTGT

841 TTCATAAGAG TCATTAGGGA TCAATAACGT TCTAATCTGT TCATAACATA CAAATTTTAT

901 GGCTGCATAG GGAAAAATTC TCAACAGGGT AGCCGAATGA CCCTGATATA GACCTGCGAC

961 ACCATCATAC CCATAGATCT GCCTGACAGC CTTAAAGAGC CCGCTAAAAG ACCCGGAAAA

1021 CCGAGAGAAC TCTGGATTAG CAGTCTGAAA AAGAATCTTC ACTCTGTCTA GTGGAGCAAT

1081 TAATGTCTTA GCGGCACTTC CTGCTACTCC GCCAGCTACT CCTGAATAGA TCACATACTG

1141 CAAAGACTGC TTGTCGATGA CCTTGGGGTT ATTTAGCTTC AAGGGCAATT TTTGGGACAT

1201 TTTGGACACA GGAGACTCAG AAACAGACAC AGAGCGTTCT GAGTCCTGGT GCTCCTGACG 1261 TAGGCCTAGA ACAGGAATTA TTGGCTTTAT TTGTTTGTCC ATTTCATAGG CTTGGGGTAA 1321 TAGATAGATG ACAGAGAAAT AGAGAAGACC TAATATTTTT TGTTCATGGC AAATCGCGGG

1381 TTCGCGGTCG GGTCACACAC GGAGAAGTAA TGAGAAGAGC TGGTAATCTG GGGTAAAAGG 1441 GTTCAAAAGA AGGTCGCCTG GTAGGGATGC AATACAAGGT TGTCTTGGAG TTTACATTGA 1501 CCAGATGATT TGGCTTTTTC TCTGTTCAAT TCACATTTTT CAGCGAGAAT CGGATTGACG 1561 GAGAAATGGC GGGGTGTGGG GTGGATAGAT GGCAGAAATG CTCGCAATCA CCGCGAAAGA 1621 AAGACTTTAT GGAATAGAAC TACTGGGTGG TGTAAGGATT ACATAGCTAG TCCAATGGAG 1681 TCCGTTGGAA AGGTAAGAAG AAGCTAAAAC CGGCTAAGTA ACTAGGGAAG AATGATCAGA 1741 CTTTGATTTG ATGAGGTCTG AAAATACTCT GCTGCTTTTT CAGTTGCTTT TTCCCTGCAA 1801 CCTATCATTT TCCTTTTCAT AAGCCTGCCT TTTCTGTTTT CACTTATATG AGTTCCGCCG 1861 AGACTTCCCC AAATTCTCTC CTGGAACATT CTCTATCGCT CTCCTTCCAA GTTGCGCCCC 1921 CTGGCACTGC CTAGTAATAT TACCACGCGA CTTATATTCA GTTCCACAAT TTCCAGTGTT

1981 CGTAGCAAAT ATCATCAGCC TACCGTTCGT ATAGCATACA TTATACGAAC GGTACTTTTT

2041 TGTAGAAATG TCTTGGTGTC CTCGTCCAAT CAGGTAGCCA TCTCTGAAAT ATCTGGCTCC

2101 GTTGCAACTC CGAACGACCT GCTGGCAACG TAAAATTCTC CGGGGTAAAA CTTAAATGTG

2161 GAGTAATGGA ACCAGAAACG TCTCTTCCCT TCTCTCTCCT TCCACCGCCC GTTACCGTCC

2221 CTAGGAAATT TTACTCTGCT GGAGAGCTTC TTCTACGGCC CCCTTGCAGC AATGCTCTTC

2281 CCAGCATTAC GTTGCGGGTA AAACGGAGGT CGTGTACCCG ACCTAGCAGC CCAGGGATGG

2341 AAAAGTCCCG GCCGTCGCTG GCAATAATAG CGGGCGGACG CATGTCATGA GATTATTGGA

2401 AACCACCAGA ATCGAATATA AAAGGCGAAC ACCTTTCCCA ATTTTGGTTT CTCCTGACCC

2461 AAAGACTTTA AATTTAATTT ATTTGTCCCT ATTTCAATCA ATTGAACAAC TATTTCGCGA

2521 AACGATGAGA TTTCCTTCAA TTTTTACTGC TGTTTTATTC GCAGCATCCT CCGCATTAGC

2581 TGCTCCAGTC AACACTACAA CAGAAGATGA AACGGCACAA ATTCCGGCTG AAGCTGTCAT

2641 CGGTTACTCA GATTTAGAAG GGGATTTCGA TGTTGCTGTT TTGCCATTTT CCAACAGCAC

2701 AAATAACGGG TTATTGTTTA TAAATACTAC TATTGCCAGC ATTGCTGCTA AAGAAGAAGG

2761 GGTATCTCTC GAGAAAAGAG AGGCTGAAGC TGAATTCGCC ACAAAACGTG GATCTCCCAA

2821 CCCTACGAGG GCGGCAGCAG TCAAGGCCGC ATTCCAGACG TCGTGGAACG CTTACCACCA 2881 TTTTGCCTTT CCCCATGACG ACCTCCACCC GGTCAGCAAC AGCTTTGATG ATGAGAGAAA 2941 CGGCTGGGGC TCGTCGGCAA TCGATGGCTT GGACACGGCT ATCCTCATGG GGGATGCCGA 3001 CATTGTGAAC ACGATCCTTC AGTATGTACC GCAGATCAAC TTCACCACGA CTGCGGTTGC

3061 CAACCAAGGC ATCTCCGTGT TCGAGACCAA CATTCGGTAC CTCGGTGGCC TGCTTTCTGC 3121 CTATGACCTG TTGCGAGGTC CTTTCAGCTC CTTGGCGACA AACCAGACCC TGGTAAACAG 3181 CCTTCTGAGG CAGGCTCAAA CACTGGCCAA CGGCCTCAAG GTTGCGTTCA CCACTCCCAG 3241 CGGTGTCCCG GACCCTACCG TCTTCTTCAA CCCTACTGTC CGGAGAAGTG GTGCATCTAG 3301 CAACAACGTC GCTGAAATTG GAAGCCTGGT GCTCGAGTGG ACACGGTTGA GCGACCTGAC 3361 GGGAAACCCG CAGTATGCCC AGCTTGCGCA GAAGGGCGAG TCGTATCTCC TGAATCCAAA 3421 GGGAAGCCCG GAGGCATGGC CTGGCCTGAT TGGAACGTTT GTCAGCACGA GCAACGGTAC 3481 CTTTCAGGAT AGCAGCGGCA GCTGGTCCGG CCTCATGGAC AGCTTCTACG AGTACCTGAT 3541 CAAGATGTAC CTGTACGACC CGGTTGCGTT TGCACACTAC AAGGATCGCT GGGTCCTTGC 3601 TGCCGACTCG ACCATTGCGC ATCTCGCCTC TCACCCGTCG ACGCGCAAGG ACTTGACCTT 3661 TTTGTCTTCG TACAACGGAC AGTCTACGTC GCCAAACTCA GGACATTTGG CCAGTTTTGC 3721 CGGTGGCAAC TTCATCTTGG GAGGCATTCT CCTGAACGAG CAAAAGTACA TTGACTTTGG 3781 AATCAAGCTT GCCAGCTCGT ACTTTGCCAC GTACAACCAG ACGGCTTCTG GAATCGGCCC 3841 CGAAGGCTTC GCGTGGGTGG ACAGCGTGAC GGGCGCCGGC GGCTCGCCGC CCTCGTCCCA

3901 GTCCGGGTTC TACTCGTCGG CAGGATTCTG GGTGACGGCA CCGTATTACA TCCTGCGGCC 3961 GGAGACGCTG GAGAGCTTGT ACTACGCATA CCGCGTCACG GGCGACTCCA AGTGGCAGGA 4021 CCTGGCGTGG GAAGCGTTCA GTGCCATTGA GGACGCATGC CGCGCCGGCA GCGCGTACTC

4081 GTCCATCAAC GACGTGACGC AGGCCAACGG CGGGGGTGCC TCTGACGATA TGGAGAGCTT 4141 CTGGTTTGCC GAGGCGCTCA AGTATGCGTA CCTGATCTTT GCGGAGGAGT CGGATGTGCA

4201 GGTGCAGGCC AACGGCGGGA ACAAATTTGT CTTTAACACG GAGGCGCACC CCTTTAGCAT 4261 CCGTTCATCA TCACGACGGG GCGGCCACCT TGCTCACGAC GAGTTGTAAT CTAGGGCGGC 4321 CGCCAGCTTG GGCCCGAACA AAAACTCATC TCAGAAGAGG ATCTGAATAG CGCCGTCGAC

4381 CATCATCATC ATCATCATTG AGTTTTAGCC TTAGACATGA CTGTTCCTCA GTTCAAGTTG 4441 GGCACTTACG AGAAGACCGG TCTTGCTAGA TTCTAATCAA GAGGATGTCA GAATGCCATT 4501 TGCCTGAGAG ATGCAGGCTT CATTTTTGAT ACTTTTTTAT TTGTAACCTA TATAGTATAG 4561 GATTTTTTTT GTCATTTTGT TTCTTCTCGT ACGAGCTTGC TCCTGATCAG CCTATCTCGC 4621 AGCTGATGAA TATCTTGTGG TAGGGGTTTG GGAAAATCAT TCGAGTTTGA TGTTTTTCTT 4681 GGTATTTCCC ACTCCTCTTC AGAGTACAGA AGATTAAGTG AGACCTTCGT TTGTGCGGAT 4741 CCCCCACACA CCATAGCTTC AAAATGTTTC TACTCCTTTT TTACTCTTCC AGATTTTCTC 4801 GGACTCCGCG CATCGCCGTA CCACTTCAAA ACACCCAAGC ACAGCATACT AAATTTCCCC 4861 TCTTTCTTCC TCTAGGGTGT CGTTAATTAC CCGTACTAAA GGTTTGGAAA AGAAAAAAGA 4921 GACCGCCTCG TTTCTTTTTC TTCGTCGAAA AAGGCAATAA AAATTTTTAT CACGTTTCTT 4981 TTTCTTGAAA ATTTTTTTTT TTGATTTTTT TCTCTTTCGA TGACCTCCCA TTGATATTTA 5041 AGTTAATAAA CGGTCTTCAA TTTCTCAAGT TTCAGTTTCA TTTTTCTTGT TCTATTACAA 5101 CTTTTTTTAC TTCTTGCTCA TTAGAAAGAA AGCATAGCAA TCTAATCTAA GGGCGGTGTT 5161 GACAATTAAT CATCGGCATA GTATATCGGC ATAGTATAAT ACGACAAGGT GAGGAACTAA 5221 ACCATGGCCA AGCCTTTGTC TCAAGAAGAA TCCACCCTCA TTGAAAGAGC AACGGCTACA 5281 ATCAACAGCA TCCCCATCTC TGAAGACTAC AGCGTCGCCA GCGCAGCTCT CTCTAGCGAC 5341 GGCCGCATCT TCACTGGTGT CAATGTATAT CATTTTACTG GGGGACCTTG TGCAGAACTC

5401 GTGGTGCTGG GCACTGCTGC TGCTGCGGCA GCTGGCAACC TGACTTGTAT CGTCGCGATC

5461 GGAAATGAGA ACAGGGGCAT CTTGAGCCCC TGCGGACGGT GCCGACAGGT GCTTCTCGAT

5521 CTGCATCCTG GGATCAAAGC CATAGTGAAG GACAGTGATG GACAGCCGAC GGCAGTTGGG

5581 ATTCGTGAAT TGCTGCCCTC TGGTTATGTG TGGGAGGGCT AAGCACTTCG TGGCCGAGGA

5641 GCAGGACTGA CACGTCCGAC GCGGCCCGAC GGGTCCGAGG CCTCGGAGAT CCGTCCCCCT

5701 TTTCCTTTGT CGATATCATG TAATTAGTTA TGTCACGCTT ACATTCACGC CCTCCCCCCA

5761 CATCCGCTCT AACCGAAAAG GAAGGAGTTA GACAACCTGA AGTCTAGGTC CCTATTTATT 5821 TTTTTATAGT TATGTTAGTA TTAAGAACGT TATTTATATT TCAAATTTTT CTTTTTTTTC 5881 TGTACAGACG CGTGTACGCA TGTAACATTA TACTGAAAAC CTTGCTTGAG AAGGTTTTGG 5941 GACGCTCGAA GGCTTTAATT TGCAAGCTGG AGACCAACAT GTGAGCAAAA GGCCAGCAAA 6001 AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CGCCCCCCTG

6061 ACGAGCATCA CAAAAATCGA CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA 6121 GATACCAGGC GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC 6181 TTACCGGATA CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CATAGCTCAC

6241 GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GTGCACGAAC

6301 CCCCCGTTCA GCCCGACCGC TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TCCAACCCGG 6361 TAAGACACGA CTTATCGCCA CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGT 6421 ATGTAGGCGG TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGAA 6481 CAGTATTTGG TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GTTGGTAGCT 6541 CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC AAGCAGCAGA 6601 TTACGCGCAG AAAAAAAGGA TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GGGTCTGACG 6661 CTCAGTGGAA CGAAAACTCA CGTTAAGGGA TTTTGGTCAT GAGATCAGAT CTAACATCCA 6721 TAATCGTATT CGCCGTTTCT GTCATTTGCG TTTTGTACGG ACCCTCACAA CAATTATCAT 6781 CTCCAAAAAT AGACTATGAT CCATTGACGC TCCGATCACT TGATTTGAAG ACTTTGGAAG 6841 CTCCTTCACA GTTGAGTCCA GGCACCGTAG AAGATAATCT TCGAAGACAA TTGGAGTTTC 6901 ATTTTCCTTA CCGCAGTTAC GAACCTTTTC CCCAACATAT TTGGCAAACG TGGAAAGTTT 6961 CTCCCTCTGA TAGTTCCTTT CCGAAAAACT TCAAAGACTT AGGTGAAAGT TGGCTGCAAA 7021 GGTCCCCAAA TTATGATCAT TTTGTGATAC CCGATGATGC AGCATGGGAA CTTATTCACC 7081 ATGAATACGA ACGTGTACCA GAAGTCTTGG AAGCTTTCCA CCTGCTACCA GAGCCCATTC 7141 TAAAGGCCGA TTTTTTCAGG TATTTGATTC TTTTTGCCCG TGGAGGACTG TATGCTGACA

7201 TGGACACTAT GTTATTAAAA CCAATAGAAT CGTGGCTGAC TTTCAATGAA ACTATTGGTG 7261 GAGTAAAAAA CAATGCTGGG TTGGTCATTG GTATTGAGGC TGATCCTGAT AGACCTGATT 7321 GGCACGACTG GTATGCTAGA AGGATACAAT TTTGCCAATG GGCAATTCAG TCCAAACGAG

7381 GACACCCAGC ACTGCGTGAA CTGATTGTAA GAGTTGTCAG CACGACTTTA CGGAAAGAGA 7441 AAAGCGGTTA CTTGAACATG GTGGAAGGAA AGGATCGTGG AAGTGATGTG ATGGACTGGA

7501 CGGGTCCAGG AATATTTACA GACACTCTAT TTGATTATAT GACTAATGTC AATACAACAG 7561 GCCACTCAGG CCAAGGAATT GGAGCTGGCT CAGCGTATTA CAATGCCTTA TCGTTGGAAG 7621 AACGTGATGC CCTCTCTGCC CGCCCGAACG GAGAGATGTT AAAAGAGAAA GTCCCAGGTA

7681 AATATGCACA GCAGGTTGTT TTATGGGAAC AATTTACCAA CCTGCGCTCC CCCAAATTAA 7741 TCGACGATAT TCTTATTCTT CCGATCACCA GCTTCAGTCC AGGGATTGGC CACAGTGGAG

7801 CTGGAGATTT GAACCATCAC CTTGCATATA TTAGGCATAC ATTTGAAGGA AGTTGGAAGG 7861 ACTAAAGAAA GCTAGAGTAA AATAGATATA GCGAGATTAG AGAATGAATA CCTTCTTCTA 7921 AGCGATCGTC CGTCATCATA GAATATCATG GACTGTATAG GTACATATAA

7981 TGATTAAACG GTCATCCAAC ATCTCGTTGA CAGATCTCTC AGTACGCGAA ATCCCTGACT 8041 ATCAAAGCAA GAACCGATGA AGAAAAAAAC AACAGTAACC CAAACACCAC AACAAACACT 8101 TTATCTTCTC CCCCCCAACA CCAATCATCA AAGAGATGTC GGAACCAAAC ACCAAGAAGC 8161 AAAAACTAAC CCCATATAAA AACATCCTGG TAGATAATGC TGGTAACCCG CTCTCCTTCC 8221 ATATTCTGGG CTACTTCACG AAGTCTGACC GGTCTCAGTT GATCAACATG ATCCTCGAAA

8281 TGGGTGGCAA GATCGTTCCA GACCTGCCTC CTCTGGTAGA TGGAGTGTTG TTTTTGACAG

8341 GGGATTACAA GTCTATTGAT GAAGATACCC TAAAGCAACT GGGGGACGTT CCAATATACA 8401 GAGACTCCTT CATCTACCAG TGTTTTGTGC ACAAGACATC TCTTCCCATT GACACTTTCC 8461 GAATTGACAA GAACGTCGAC TTGGCTCAAG ATTTGATCAA TAGGGCCCTT CAAGAGTCTG 8521 TGGATCATGT CACTTCTGCC AGCACAGCTG CAGCTGCTGC TGTTGTTGTC GCTACCAACG 8581 GCCTGTCTTC TAAACCAGAC GCTCGTACTA GCAAAATACA GTTCACTCCC GAAGAAGATC 8641 GTTTTATTCT TGACTTTGTT AGGAGAAATC CTAAACGAAG AAACACACAT CAACTGTACA 8701 CTGAGCTCGC TCAGCACATG AAAAACCATA CGAATCATTC TATCCGCCAC AGATTTCGTC 8761 GTAATCTTTC CGCTCAACTT GATTGGGTTT ATGATATCGA TCCATTGACC AACCAACCTC

8821 GAAAAGATGA AAACGGGAAC TACATCAAGG TACAAGATCT TCCACAAGGA ATTCGTGGTC 8881 ATTATTCTGC CCAAGATGAT TACAATTTGT GTTTATCGGT TCAACCTTTC ATTGAATCTG 8941 TAGATGAGAC AACAGGCCAA GAATTTTTCA AACCTCTGAA AGGTGTATTT GATGACTTGG 9001 AATCTCGCTT TCCTCACCAT ACAAAGACTT CCTGGAGAGA CAGATTCAGA AAGTTTGCCT

9061 CTAAATACGG TGTTCGTCAG TACATCGCGT ATTATGAAAA GACTGTTGAA CTCAATGGTG 9121 TTCCTAATCC GATGACGAAC TTTACCTCAA AGGCTTCCAT TGAAAAATTT AGAGAAAGAC 9181 GCGGGACTTC ACGTAACAGT GGCCTTCCAG GCCCGGTTGG TGTAGAAGCT GTAAGCTCTT

9241 TGGACCACAT ATCCCCATTG GTCACATCTA ATTCCAATTC TGCAGCTGCT GCAGCTGCTG

9301 CCGCAGCAGT TGCAGCCTCT GCCTCTGCTT CTTCAGCTCC TAATACTTCA ACTACCAATT 9361 TCTTTGAACA GGAGAATATT GCCCAAGTTC TCTCTGCACA TAACAACGAG CAGTCTATTG 9421 CAGAAGTTAT TGAGTCCGCA CAGAATGTCA ACACCCATGA AAGTGAACCT ATAGCTGATC 9481 ATGTTCGAAA AAATCTTACA GACGATGAAT TGCTTGACAA AATGGATGAT ATTTTAAGCT 9541 CCAGAAGTCT AGGCGGACTA GATGACTTGA TAAAGATCCT CTACACTGAG CTGGGATTTG 9601 CTCATCGTTA TACCGAATTT CTTTTTACCT CATGTTCTGG TGATGTGATT TTCTTCCGAC 9661 CATTAGTGGA ACATTTCCTT CTTACTGGTG AGTGGGAGCT GGAGAATACT CGTGGCATCT 9721 GGACCGGTCG TCAAGACGAA ATGCTACGTG CTAGCAATCT AGATGACCTG CACAAGTTAA 9781 TTGACCTGCA TGGGAAAGAA CGTGTTGAGA CCAGAAGAAA AGCCATCAAG GGAGAATGAT 9841 CATAAGAAAT GAAAAACGTA TAAGT

Table 2. SEQ ID NO: 2.

(M) AKADGSLLY YNPH NPPRRY YFYMAI FAVS VICVLYGPSQ

QLSSPKI DYD PLTLRSLDLK TLEAPSQLSP GTVEDN LRRQ

LEFH FPYRSY EPFPQHIWQT WKVSPSDSSF PKNFKDLGES

WLQRSPNYDH FVI PDDAAWE LI H H EYERVP EVLEAFHLLP

EPILKADFFR YLI LFARGGL YADMDTM LLK PI ESWLTFNE

TIGGVKN NAG LVIGI EADPD RPDWH DWYAR RIQFCQWAIQ

SKRG HPALRE LIVRVVSTTL RKEKSGYLNM VEG KDRGSDV

MDWTGPG I FT DTLFDYMTNV NTTGHSGQGI GAGSAYYNAL

SLEERDALSA RPNGEM LKEK VPG KYAQQVV LWEQFTNLRS

PKLI DDILI L PITSFSPG IG HSGAG DLNHH LAYIRHTFEG

SWKD

Table 3.

Table 3. continued

Table 3. continued

Table 3. continued

Table 3. continued

Table 4. SEQ ID NO: 53.

1 AACGTCAAAG ACAGCAATGG AGTC TATT 6ATAACACCA CTGGCA6AGC GGTTCGTACG 61 TCGTTTTGGA GCCGATATGA S¾CTCAGCGT GCTAACAGCA C6ATTGACAA G GACTCT 121 GAGTGACAGT AGGTTGAGTA AAGTATTCGC TTAGATTCCC AACCTTCGTT TTATTCTTTC 131 GTAGACAAAG AAGCTGCATG CGAACATAGG GACAACTTTT ATAAATCCAA TTGTCAAACC 241 AACGTAAAAC CCTCTGGCAC CATTTTCAAC ATATATTTGT GAAGCAGTAC 6CAATATCGA 381 TAAATACTCA CCSTTSTTTG TAACAGCCCC AACTTGCATA CGCCTTCTAA TGACCTAAA 361 TGGATAAGCC GCAGCTTGT6 CTAACATACC AGCAGCACCG CCCGCGGTCA GCTGCGCCCA

421 CACATATAAA GGCAATCTAC GATCATGGGA GGAATTAGTT TTGACCGTCA GGTCTTCAAG 481 AGTTTTGAAC TGTTCTTCTT GAACTGTGTA ACCTTTTAAA T6ACGGGATC TAAATACGTC 541 ATGGATGAGA TCATGTGTGT AAAAACTGAC TCCAGCATAT GGAATCATTC CAAAGATTGT 681 A6GAGCGAAC CCACGATAAA AGTTTCCCAA CCTTGCCAAA GTGTCTAATG CTGTGACTTG 661 AAATCTGGGT TCCTCGTTGA AGACCCTGCG TA TATGCCC AAAAACTTTC CTCCAC6AGC 721 CCTATTAACT TCTCTATGAG TTTCAAATGC CAAACGGACA CGGATTAGGT CCAATGGGTA 781 AGTGAAAAAC ACA6AGCAAA CCCCAGCTM TGAGCCGGCC AGTAACCGTC TTGGAGCTGT 841 TTCATAAGAG TCATTAGGGA TCAATAACGT TCTAATCTGT TCATAACATA CAAATTTTAT 991 GGCTGCATAG GGAAAAATTC TCAACAGGGT AGCCGAATCA CCCT6ATATA GACCTGCGAC 961 ACCATCAT C CCATAGATCT GCCTGACAGC OTAAAGAGC CCGCTAAAAG ACCCGGA AA 1021 CC6AGAGAAC TCTGGATTAG CAGTCTGAAA AAGAATCTTC ACTCTGTCTA GTGGAGCAAT 1081 TAATGTCTTA GCGGCACTTC CTGCTACTCC GCCAGCTACT CCTGAATAGA TCACATACTG 1141 CAAAGACTGC TTGTC6AT6A CCTTGGGGTT ATTTAGCT7C AAGGGCAATT TTTGGGACAT 1281 TTTGCiACACA GGAGACTCA6 AAACAGACAC AGAGCGTTCT GAGRXTGGT 6CTCCT6AC6 1261 TAGGCCTAGA ACAGGAATTA TTGGCTTTAT TTGTTTGTCC ATTTCATAGG CTTGGGGT 1321 TAGATAGATG ACAGAGAAAT AGAGAAGACC TAATATTTTT TGTTCATGGC AAATCGCGGG 1381 TTCGCGGTCG GGTCACACAC GGAGAAGTAA TGAGAAGAGC: TGGTAATCTG GGGTAAAAGG 1441 GTTCAAAAGA AGGTCGCCTG GTAGGGATGC AATACAAGGT TGTCTTGGAG TTTACATTGA 1591 CCAGATGATT TGGCTTTTTC TCTGTTCAAT TCACATTTTT GAGCGAGAAT CGGATTGACG 1561 GAGAAATGGC GGGGTGTGGG GTGGATAGAT GGCAGAAATG CTCGCAATCA CCGCGAAAGA 1621 AAGACTTTAT GGAATAGAAC TACTGGGTGG TGTAAGGATT ACATAGCTAG TCCAATGGAG 1681 TCCGTTGGAA AGGTAAGAAG AAGCTAAAA CGGCTAAGTA ACTAG6GAAG AATGATCAGA 1741 CTTT6ATTTG ATGAGGTCTG AAAATACTCT GCTGCTTTTT CAGTTGCTTT TTCCCTGCAA 1881 CCTATCATTT TCCTTTTCAT AAGCCTGCCT TTTCTGTTTT CACTTATATG AGTTCCGCCG 1861 AGACTTCCCC AAATTCTCTC CTGGAACATT CTCTATCGCT CT CTTCCAA GTTGCGCCiX 1921 CTGGCACTGC CTAGTAATAT TACCACGCGA CTTATATTCA GTTCCACAAT TTCCAGTGTT 1981 CGTAGCAAAT ATCATCAGCC ATGGCGAAGG CAGATGGCAG TTTGCTCTAC TATAATCCTC 2841 ACAATCCACC CAGAAGGTAT TACTTCTACA TGGCTATATT CGCCGTTTCT GTCATTTGCG 2181 TTTTGTACGG ACCCTCACAA CAATTA CAT aCCAAAAAT AGACTATGAT CCATTGACGC 2161 TCCGATCACT TGATTTGAAG ACTTTGGAAG CTCCTTCACA GTTGAGTCCA GGCACCGTAG 2221 AAGATAATCT TCGoagocoa ttggagtttc attttcctta ccgcogtt c gaoccttttc 2281 cccoacotot ttgataaocQ tggaaagttt c ccctctga tagttccttt ccgaaaeact 2341 tcaaogactt Qggtgoaagt tggctgcaaa ggtccccaaa ttatgcrtccr tttgtgatac 2431 ccgotgotgc ogcatgggao cttattaicc atgaatacga acgtg acca go gtcttgg 2461 aagctctoga tgctcocqgc aatgctgtta aggttcgtat ggagaoactg ggoc tottt 2521 oattatttag agattttaae ttacatttag attcgataga tccacaggac gggtgtggtc 25S1 gceatgateg cgtogtcgot agtggctcca agtagcgaag cgagcaggac tgtgcggcgg 2641 ccocagcggt cggaeagtgc tccgagaacg ggtgcgcata gaaattgcat coacgcotat. 2781 egcgctagca gcacgccoto gtgoctggcg atgctgtcgg aatggacg t otcccgcaag 2761 oggcecggca gta cggcat aaccaagcc atgcctacag catccagggt gacggtgccg 2821 oggotgecga tgaocgcatt gttagatttc atacacggtg cctgactgcg t agcaottt 2881 aaetgtgata oactaccgca ttaaagctga tcttttttgt agaaotgtct tggtgtcctc 2941 gtccaatcag jjtagccatct ctgaaatatc tggctccgtt gcaactccga acgacctgct 3081 ggcaocgtoa oattctccgg ggtaaaactt aaatgtggag taatggaocc agoaacgtct 3861 cttcccttct ctctccttcc accgcccgtt accgtcccta ggaaatttta ctctgctggo 3121 gagcttcttc taeggcccec ttg ag aat gctcttccca gcattacgtt gcgggtaaa 3181 cggoggtc t gtacccgocc tagcagccca gggatggaaa agt ccggcc gtcgctggco 3241 ataatagcgg gcgga gcot gtcatgagat tottggaaac caccogaotc gaatataaaa 3301 ggcgaacacc tttccawtt ttggtttctc ctgoccoaaa gacttta ot ttaotttatt 3361 tgt CEictott tcootcoatt gaacaactat ttcg gaaac gatgogattt ccttcaattt 3421 ttactgctgt tttattcgco gcatcctccg cat agctgc tccagtcaoc actacaacag 3481 oogatgoooc ggcacoaott ccggctgaag ctgtcatcgg ttactcagot ttagaagggg 3541 otttcgotgt tgctgttttg ccottttcca acagcacaaa taacgggtta ttgtttotaa 3681 atoctactot tgccagcatt gctgctoaag aogaaggggt atctctcgetg eaaogagagg Table 4. continued

3661 ctgaagctga attcgccaco aaacgtggat ctcccaaccc tacgagggcg gcagcagtca 3721 aggccgcatt ccogaegteg tggaaegctt accaccattt tgectttccc catga gacc 37S1 tccacccggt cagcaacagc tttgatgatg ogagaaacgg ctggggctcg tcggcaatcg 3841 otggcttgga cacggctatc ctcatggggg atgccgacat tgtgaacacg atccttcagt 3901 otgtocegco gatcaa ttc accaegattg cggttgccaa ccaaggotcc tccgtgttcg 3961 agaccaacat teggtocctc ggtggcctgc tttctgccta tgacctgttg cgaggtcctt 4821 tcogctc tt Qgcgacaaac cagaccctgg taaacagcct tctgaggcog gctcaaacac 4081 tggccao gg cctcaoggtt gegttcacca ctcccagcgg tgtcccggoc cctaccgtct 4141 tcttcaoccc tactgtccgg agaagtggtg catctagcaa caacgtcgct gaaa tggaa 4201 gcctggtgct gagtggaco cggttgagcg acctgacggg aoacccgcog totgcccagc 4261 ttgcgcagaa gggcgogtcg tatctcctga otccaoaggg aageccggag gcatggcctg 4321 gcctgattgg oocgtttgtc agcacgagca acggtacctt tcaggatagc agcggcagct 4381 ggtccggcct cotggocogc ttctaegagt acctgatcaa gatgtocctg tacgacccgg 4441 ttgcgtttgc acoctacoctg gatcgctggg tccttggtgc cgactcgacc attgggcatc 4501 tcggctctea cccgtcgocg ogcaaggact tgaccttt t gtcttcgtoc oocggocagt 4561 ctacgtcgcc aoactcagga catttggcca gttttggcgg tggcciacttc atcttgggag 4621 gcattctcct gaacgagcao aagtacattg actttggaat caagcttgcc agctcgtoct 4681 ttgg ctcgta cacccagacg gcttctggaa tcggcccc a aggcttcgcg tgggtggaca 4741 gcgtgacggg cgccggcggc tcgccgccct cgtcccagtc cgggttctoc tcgtcggcag 4S01 gattctgggt gacggcaccg tattacatcc tgcggccgga gacgctggog agct gtact 4861 Qcgcataccg egtCQcgggc gactccaagt ggcaggacct gg gtgggoa gcg tgagtg 4921 ccattgaggo cgcat§cc§c gccggcagcg cgtactcgtc catcaacgac gtgacgcogg 4981 ccQOCggcgg gggtgcctct gacgatatgg agagcttctg gtttgccgog gcgctcaagt 5041 atgcgtacct gatctttgeg gaggagtcgg atgtgcaggt gcaggccacc ggcgggaaca 5181 aatttgtctt taacaeggag gcgcacccct ttagcotccg ttcatcatca cgacggggcg 5161 gccoccttgc t acgacgag ttgtaatctn gggcGGCCGC CAGCTT&GGC CCGMCAAAA 5221 ACTCATCTCA GAAGAGGATC TGAATAGCGC CGTCGACCAT CATCATCATC ATCATT&AGT 5281 TTTAGCCTTA GACATGACTG TTCCTCAGTT CAAGTTGGGC ACTTAC6AGA A6ACCGGTCT 5341 TGCTAGATTC TAAT AAGAG GATGTCA6AA TGCCATTTGC CTGAGAGATG CAGGCTT AT 5481 TTTTGATACT TTTTTATTT ^' G TAACCTATAT AGTATAGGAT TTTTTTTGTC ATTTTGTTTC 5461 TTCTCGTACG AGCTTGCTCC TGATCAGCCT ATC7CGCAGC TGATGAATAT C77G76G7AG 5521 GGGTTTGGGA AAATCATTCG AGTTTGATGT 7TT7CT7GG7 ATT CCCACT CCTCTTCAGA 5581 GTACAGAAGA TTAAGTGAGA CC7TCGT7TG 7GCGGATCCC CCACACACCA TAGC7TCAM 5641 ATGTTTCTAC TGITTTTTTA C7CT7CCAGA TTTTCTCGGA CTCCGCGCAT CGCCGTACCA 5701 CTTCAAAACA CCCAAGCACA GCAT CTAAA TTTCCCCTCT TTCTTCCTCT AG&GT&TCST 5761 TAATTACCCG TACTAAAGGT TTGGAAAAGA AAMAGAGAC CGCCTCG7TT CTTTTTCTTC 5821 GTCGAAAAAG GCAATAAAAA TTTTTAT AC GTT7CT7T77 C7T6AAAAT7 T77T77T7TG 5881 ATTTTTTTCT CTTTCGATGA CCTCCCATTG ATATTTMGT TMTAAACGG TCTTCAATTT 5941 CTCAAGTTTC AGTTTCATTT T7CT7GT7CT AT7ACAAC7T TT7TTACTTC 7TGC7CAT7A 60S! GAAAGAAAGC ATAGCAATCT AATCTAAGGG CGGTG7TGAC M7TMTCAT CGGCATA67A 6061 TATCGGCATA 6TATAATACG ACAAGG7GAG GAAC7MACC A7GGCCMGC CTTT&TCTCA 6121 AGAAGAATCC ACC TCATTG AAAGAGCAAC GGCTACAATC AACAGCA7CC CCATCTC7GA 6181 AGACTACAGC GTCGCCAGCG CAGCTCTCTC TAGCGACGGC CGCATCTTCA CTGGTGTCAA 6241 TGTATATCAT TTTACTGGGG GACCTTGTGC AGAAGTGGTG GTGCTGGGCA C7GC7GCTGC 6301 TGCGGCAGCT GGCAACCTGA OTG7ATCGT CGCGATCGGA AATGAGAACA GGGGCATCTT 6361 GAGCCCCTGC GGACGGTGCC GACAGGTGCT TCTCGA7CTG CA7CCTG6GA TCAAA6CCA7 6421 AGTGAAGGAC AGTGATGGAC AGCCGAC GC AGTTGGGATT GGTGAATTGC T&CCCTC7GG 6481 TTATGT6TG6 GAGGGCTAAG CACTTCGTGG CGGAGGAGCA GGACTGACAC GTCC&ACGCG 6541 GCCCGACGGG TCCGAGGCCT CGGAGATCCG TCCCCCT7TT CC7TTGT GA Tatcatgtoa 6601 ttagttatgt cacgcttaca ttcacgccct ccccccaca ccgctctaoc cgaaaaggaa 6661 ggogttogoc aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta 6721 og Qcgttat ttatotttco aatttttctt ttttttctgt acagacgcgt gtacgcotgt 6781 a tttatac tgaoaocctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc 6841 oogctggogQ ccaacctgtg agcaaaaggc cagcaaaagg ccaggaoccg taaoaaggcc Table 4. continued

6901 gcgttgctgg cgtttttcca taggctccgc ccccctgocg agcatcacaa aaatcgacgc

6961 tcoagtcago ggtggcgaaa ccegacagga ctatoaagot accaggcgtt tccccctgga

7021 agctccctcg tgcgctctcc tgttccgacc ctgccgctto ccggatocct gtc gccttt

7681 ctcccttcgg goagcgtggc gctttctcao tcctcacgct gtaggtatct ogttcggtg

7141 taggtcgttc gctccaagct ggg tgtgtg cacgoacccc ccottcagcc cgoccgctgc

7201 gccttatccg gtooctotcg tcttgagtcc oocecggtoo gocaegoctt atcgccactg

7261 gcagcagcca ctggtaacog gattagcaga gcgaggtatg taggcggtcc tacagagttc

7321 ttgaogtggt ggcctoaeta eggctocaet agooggocag tatttggtot ctgcgctctg

7331 ctgaagccag ttaccttcgg aaaaagagtt gctogctctt gotccggcoo acooaccocc

7441 gctggtogcg gtggtttttt tgtttgcoag cogeogcrttG cgcgcaga a a aaggatct

7561 caagaagatc ctttgatctt ttctacgggg tctcacgctc agtgcaocca oaactcacgt

7561 to ggg ttt tggtcotgog atcagotcto acotccotao tcctott gc gtttctgtc

7621 Qtttgcgt t tgtQcggacc ctcocaacaa ttatcatctc ooaootago ctatgatcca

7681 ttgacgctce gatcacttga tttgoagoct ttgcQogctiz cttcacogtt gagtccoggc

7741 occgtagoag o aatcttCG AAGACAATTG GAGTTTCATT TTCCTTACCG CAGTTACGAA

7801 Cx I I I ! CCCC AACATATTT6 GCAAACGTGG AAAGTTTCTC CCTCTGATAG TTCCTTTCCG

7861 AAAAACTTCA AA6ACTTAG6 TGAAAGTTGG CTGCAAAGGT CCCCAAATTA TGATCATTTT

7921 GTGATACCCG ATGATGCAGC ATGG6AACTT ATTCACCATG AATACGAACG TGTACCAGAA

7981 GTCTTGGAAG CTTTCCACCT 6CTACCAGAG CCCATTCTAA AGGCCGATTT TTTCAGGTAT

8041 TTGATTCTTT TTGCCCGTG6 AGGACTGTAT GCTGACATGG ACACTATGTT ATTAAAACCA

8181 ATAGAATCGT GGCTGACTTT CAATGAAACT ATTGGTGGAG TAAAAAACAA TGCTGGGTTG

S161 GTCATTGGTA TTGAGGCTGA TCCT6ATA6A CCTGATTGGC ACGACTGGTA TGCTAGAAGG

8221 ATACAATTTT 6CCAATG6GC AATTCAGTCC AAACGAGGAC ACCCAGCACT GCGTGAAOG

8281 ATTGTAAGAG TTGTCAGCAC GACTTTACGG AAAGAGAAAA GCGGTTACTT GAACATGGTG

8341 GAAGGAAAGG ATCGTG6AA6 TGATGT6ATG GACTGGACGG GTCCAGGAAT ATTTACAGAC

S401 ACTCTATTTG ATTATAT6AC TAAT6TCAAT ACAACAGGCC ACTCAGGCCA AGGAATTGGA

8461 GCTGGCTCAG CGTATTACAA TGCCTTATCG TTGGAAGAAC GTGATGCCCT CTCTGCKGC

8521 CCGAACGGAG AGATGTTAAA AGAGAAAGTC CCAGGTAAAT ATGCACAGCA GGTTGTTTTA

8581 TGGGAACAAT TTACCAACCT GCGCTCCCCC AAATTAATCG ACGATATTCT TATTCTTCCG

S641 ATCACCAGCT TCAGTCCAGG 6ATT6GCCAC AGTGGAGCTG GAGATTTGAA CCATCACCTT

8701 GCATATATTA 6GCATACATT TGAAGGAAGT TGGAAGGACT AAAGAAAGCT AGAGTAAAAT

8761 AGATATAGCG AGATTA6AGA ATGAATACCT TGTTCTAAGG GATCGTCCGT CATGATAGAA

8821 TATCATGGAC TGTATAGTTT T■ ITT■ TGTA CATATAATGA TTAAACGGTC ATCCAACATC

8881 TC6TTGACAG ATCTCTCAGT ACGCGAAATC CCTGACTATC AAAGGAAGAA CCGATGAAGA

8941 AAAAAACAAC AGTAACCCAA ACACCACAAC AAACACTTTA TCTTCTCCCC CCCAACACCA

9081 ATCATCAAAG AGATGTCGGA ACCAAACACC AAGAAGCAAA AACTAACCCC ATATAAAAAC

9861 ATCCTGGTA6 ATAATGCTGG TAACCCGCTC TCCTTCCATA TTCTGGCCTA GTTCACGAAG

9121 TCTGACCGGT CTCAGTTGAT CAACATGATC CTCGAAATGG GTGGGAAGAT CGTTCGAGAG

9181 CTGCCTCCTC TGGTAGATGG AGTGTTGTTT TTGACAGCGG ATTACAAGTC TATTGATGAA

9241 GATACCCTAA AGCAACTGGG GGACGTTCCA ATATACAGAC ACTCCTTCAT CTACCAGTGT

9361 TTTGTGCACA AGACATCTCT TCCCATTGAC ACTTTCCGAA TTGACAAGAA CGTCGACTTG

9361 GCTCAAGATT TGATCAATAG GGCCCTTCAA GAGTCTGTGG ATCATGTCAG TTCTGCCAGC

9421 ACAGCTGCAG CTGCTGCTGT TGTTGTCGCT ACCAACGGCC TGTCTTCTAA ACCAGACGCT

9481 CGTACTAGCA AAATACAGTT CACTCCCGAA GAAGATCGTT TTATTCTTGA GTTTGTTAGG

9541 AGAAATCCTA AACGAAGAAA CACACATCAA CTGTACACTG AGCTCGCTCA GCACATGAAA

9601 AACCATACGA ATCATTCTAT CCGCCACAGA TTTCGTCCTA ATCTTTCCCC TCAACTTGAT

9661 TGGGTTTATG ATATCGATCC ATTGACCAAC CAACCTCGAA AAGATGAAAA CGGGAACTAC

9721 ATCAAGGTAC AAGATCTTCC ACAA6GAATT CGTGGTCATT ATTCTGCCCA AGATGATTAC

9781 AATTTGTGTT TATC6GTTCA ACCTTTCATT GAATCTGTAG ATGAGACAAC AGGCCAAGAA

9841 TTTTTCAAAC CTCTGAAAG6 TGTATTTGAT GACTTGGAAT CTCGCTTTCC TCACCATACA

9901 AAGACTTCCT GGAGAGACA6 ATTCAGAAAG TTTGCCTCTA AATACGGTGT TCGTCAGTAC

9961 ATCGCGTATT ATGAAAAGAC TGTT6AACTC AATGGTGTTC CTAATCCGAT GACGAACTTT

10021 ACCTCAAAGG CTTCCATTGA AAAATTTAGA GAAAGACGCG GGACTTCACG TAACAGTGGC

10081 CTTCCAGGCC CGGTTGGTGT AGAAGCTGTA AGCTCTTTGG ACCACATATC CCCATTGGTC

10141 ACATCTAATT GCAATTCTGC AGCTGCTGCA GCTGOGCCG CAGCAGTTGC AGCCTCTGCC

10291 TCTGCTTCTT CAGCTCCTAA TACTTCAACT ACCAATTTO TTGAACAGGA GAATATTGCC

10261 CAA6TTCTCT CTGCACATAA CAACGAGCAG TCTATTGCAG AAGTTATTGA GTCCGCACAG

10321 AATGTCAACA CCCATGAAAG TGAACCTATA GCTGATCATG TTCGAAAAAA TCTTACAGAC

18381 6ATGAATT6C TTGACAAAAT GGATGATATT TTAAGCTC A GAAGTCTAGG CGGACTAGAT

10441 6ACTTGATAA AGATCCTCTA CACTGAGOG GGATTTGCTC ATCGTTATAC CGAATTTCTT

10531 TTTACCTCAT GTTCTGGTGA TGTGATTTTC TT CGA CAT TAGTGGAACA TTTCCTTCTT

10561 ACTGGTGAGT GGGAGCTGGA GAATAOCGT GGCATCTGGA CCGGTCGTCA AGACGAAATG

10621 CTACGTGCTA GCAATCTAGA TGACCTGCAC AAGTTAATTG AtCTGCATGG GAAAGAACGT

10681 GTTGAGACCA GAAGAA ACC CATCAAGGGA GAATGATCAT AAGAAATGAA AAACGTATAA

10741 GT Table 5. SEQ ID NO: 54 (top) and SEQ ID NO: 55 (bottom) AMINO ACID SEQUENCE AKADGSLLY YNPH NPPRRY YFY AI FAVS VICVLYGPSQ QLSSPKI DYD PLTLRSLDLK TLEAPSQLSP GTVEDNLRRQ LEFHFPYRSY EPFPQHIWQT WKVSPSDSSF PKN FKDLGES WLQRSPNYDH FVI PDDAAWE LI HH EYERVP EVLEALDAH R NAVKVRM EKL G LI

DNA SEQUENCE

ATGGCGAAGG CAGATGGCAG TTTGCTCTAC TATAATCCTC ACAATCCACC CAGAAGGTAT TACTTCTACA TGGCTATATT CGCCGTTTCT GTCATTTGCG TTTTGTACGG ACCCTCACAA CAATTATCAT CTCCAAAAAT AGACTATGAT CCATTGACGC TCCGATCACT TGATTTGAAG ACTTTGGAAG CTCCTTCACA GTTGAGTCCA GGCACCGTAG AAGATAATCT TCGAAG ACAA TTGGAGTTTC ATTTTCCTTA CCGCAGTTAC GAACCTTTTC CCCAACATAT TTGGCAAACG TGGAAAGTTT CTCCCTCTGA TAGTTCCTTT CCGAAAAACT TCAAAGACTT AGGTGAAAGT TGGCTGCAAA GGTCCCCAAA TTATGATCAT TTTGTGATAC CCGATGATGC AGCATGGGAA CTTATTCACC ATGAATACGA ACGTGTACCA GAAGTCTTGG AAGCTCTAGA TGCTCACCGC AATGCTGTTA AGGTTCGTAT GGAGAAACTG GGACTTATTT AA

Table 6. SEQ ID NO: 56 (top) and SEQ ID NO: 57 (bottom) AMINO ACID SEQUENCE

M RSDLTSI IV FAVSVICVLY GPSQQLSSPK I DYDPLTLRS LDLKTLEAPS QLSPGTVEDN LRRQLEFHFP YRSYEPFPQH IWQTWKVSPS DSSFPKNFKD LGESWLQRSP NYDHFVI PDD AAWEU HHEY ERVPEVLEAF H LLPEPI LKA DFFRYLI LFA RGGLYADM DT M LLKPI ESWL TFN ETIGGVK N NAG LVIGI E ADPDRPDWH D WYARRIQFCQ WAIQSKRGHP ALRELIVRVV STTLRKEKSG YLN VEG KDR GSDVM DWTG P GIFTDTLFDY MTNVNTTG HS GQG IGAGSAY YNALSLEERD ALSARPNG EM LKEKVPG KYA QQVVLWEQFT N LRSPKLI DD ILI LPITSFS PG IGHSGAG D LNHHLAYIRH TFEGSWKD

DMA SEQUENCE

1 atgagatcag atctaacatc cataatcgta ttcgccgttt ctgtcatttg cgttttgtac

61 ggaccctcac aacaattatc atctccaaaa atagactatg atccattgac gctccgatca

121 cttgatttga agactttgga agctccttca cagttgagtc caggcaccgt agaagataat

1S1 CTTCGAAGAC AATTGGAGTT TCATTTTCCT TACCGCAGTT ACGAACCTTT TCCCCAACAT 241 ATTTGGCAAA CGTGGAAAGT TTCTCCCTCT GATAGTTCCT TTCCGAAAAA CTTCAAAGAC 301 TTAG6TG AAA GTTGGCTGCA AA6GTCCCCA AATTATG ATC ATTTTGTGAT ACCCGATG AT 361 GCAGCATGGG AACTTATTCA CCATGAATAC GAACGTGTAC CAGAAGTCTT GGAAGCTTTC 421 CACCTGCTAC CAGAGCCCAT TCTAAAGGCC GATTTTTTCA GGTATTTGAT TCTTTTTGCC 481 CGTGGAGGAC TGTATGCTGA CATGGACACT ATGTTATTAA AACCAATAGA ATCGTGGCTG 541 ACTTTCAATG AAACTATTGG TGGAGTAAAA AACAATGCTG GGTTGGTCAT TGGTATTGAG 601 GCTGATCCTG ATAGACCTGA TTGGCACGAC TGGTATGCTA GAAGGATACA ATTTTGCCAA 661 TGGGCAATTC AGTCCAAACG AGGACACCCA GCACTGCGTG AACTGATTGT AAGAGTTGTC 721 AGCACGACTT TACGGAAAGA GAAAAGCGGT TACTTGAACA TGGTGGAAGG AAAGGATCGl 781 GGAAGTGATG TGATGGACTG GACGGGTCCA GGAATATTTA CAGACACTCT ATTTGATTAT 841 ATGACTAATG TCAATACAAC AGGCCACTCA GGCCAAGGAA TTGGAGCTGG CTCAGCGTAT 901 TACAATGCCT TATCGTTGGA AGAACGTGAT GCCCTCTCTG CCCGCCCGAA CGGAGAGATG 961 TTAAAAGAGA AAGTCCCAGG TAAATATGCA CAGCAGGTTG TTTTATGGGA ACAATTTACC 1021 AACCTGCGCT CCCCCAAATT AATCGACGAT ATTCTTATTC TTCCGATCAC CAGCTTCAGT 1081 CCAGGGATTG GCCACAGTGG AGCTGGAGAT TTGAACCATC ACCTTGCATA TATTAGGCAT 1141 ACATTTGAAG GAAGTTGGAA GGACTAA