FIELD OF THE INVENTION

The present invention relates to novel protein disulfide redox agents, DNA sequences expressing these agents, and methods for their production.

The invention furthermore relates to compositions comprising

(i) a protein disulfide redox agent in combination with (ii) at least one redox partner, and optionally (iii) at least one or more other enzymes. The compositions can be used for the treatment or degradation of scleroproteins, especially hair, skin and wool, treatment and cleaning of fabrics, as additives to detergents, thickening and gelation of food and fodder, and pharmaceuticals for the alleviation of eye sufferings.

BACKGROUND OF THE INVENTION

The use of protein disulfide redox agents such as protein disulfide reductases, protein disulfide isomerases, protein disulfide oxidases, protein disulfide oxidoreductase, protein disulfide transhydrogenases, sulfhydryl oxidase, and thioredoxins for various purposes has been known for some time.

Protein disulfide redox agents catalyses the general reaction:

R-L_SH + R ₂ -SH + Enz _oχ < ^■ * R ₁ -S-S-R ₂ + Enz _red (reaction I)

where R-^ and R ₂ represent protein entities which are the same or different, either within the same polypeptide or in two polypeptides, Enz _oχ is a protein disulfide redox agent in the oxidised state, and Enz _recj is a protein disulfide redox agent in the reduced state. EC 5.3.4.1 (Enzyme Nomenclature, Academic Press, Inc., 1992) refers to an enzyme capable of catalysing the rearrangement of -S-S- bonds in proteins and EC 1.6.4.4 and

EC 1.8.4.2 is an example of enzymes catalysing the reaction with NAD(P)H and glutathione as a mediator, respectively.

This type of activity has in the past been designated as e.g. protein disulfide isomerase, protein disulfide oxidase, protein sulfhydryl oxidase, protein disulfide reductase, sulfhydryl isomerase, disulfide isomerase, protein disulfide transhy- drogenase, protein disulfide oxidoreductase and sulfhydryl oxidase.

The uses of such enzymes have all been connected with reduction of protein disulfide linkages to free protein sulhydryl groups and/or the oxidation of protein sylfhydryl groups to protein disulfide linkages, and/or the rearrangement of disulfide linkages in the same or between different polypeptides, and sometimes with both these processes in sequence.

The protein disulfide redox agents of this invention can be divided into four main groups of enzymes, thioredoxin type (TRX) , protein disulfide isomerase type (PDI) , disulfide Bond Formation protein type (dsbA) and protein engineered deriva¬ tives, chemical modifications and hybrids of TRX and/or PDI and/or dsbA (ENG, sometimes also designated variants or muteins of TRX, PDI or dsbA) .

TRX is a 12-kDa protein having a redox-active disulfide/dithiol and catalysing thiol-disulfide exchange reactions (Edman et al., Nature 317:267-270, 1985; Holmgren, Annu. Rev. Biochem. 54:237-271, 1985; Holmgren, J. Biol. Chem. 264:13963-13966, 1989) .

PDI consists of two subunits, each consisting of two domains which are homologous to TRX.

DsbA is a 21-kDa protein known to be capable of reducing disulfide bonds of insulin and activity common to disulfide oxidoreductases (Bardwell et al. , Cell, Vol. 67, 581-589, 1991) .

TRX, DsbA and the two domains in the subunits of PDI generally comprise a sequence which may be represented as below:

R ¹ -Cys-X-Y-Cys-R ² For TRX and PDI are R ¹ and R ² each different amino acid sequences, X generally is Gly, and Y generally is Pro or His, respectively.

TRX from the T ₄ -bacteriophage has the sequence:

R ₁ -Cys-Val-Tyr-Cys-R ₂

DsbA from E. coll has generally the sequence:

R ₁ -Cys-Pro-His-Cys-R ₂

In the context of this invention a protein disulfide redox agent may therefore be defined as an enzyme exhibiting the above sequence, but where X and Y can be any amino acid residue, and catalysing reaction I above.

ENG can be prepared by a variety of methods based on standard recombinant DNA technology:

1) by using site-directed or random mutagenesis to modify the genes encoding TRX, dsbA or PDI in order to obtain ENG with one or more amino acid changes, such as replacements, insertions, and/or deletions,

2) by inhibiting or otherwise avoiding dimerisation of the subunits of PDI, thus giving rise to PDI monomers,

3) by producing partial monomers of PDI, dsbA or TRX, in which regions of the NH2- or COOH termini of PDI, dsbA or TRX are lacking,

4) by creating hybrids of PDI, dsbA, TRX and/or ENG,

5) by chemically or enzymatically modifying the products of 1) - 4) , and 6) by a combination of any of 1) -5) .

ENG preparation by standard recombination DNA technology for TRX and PDI according to 1) was described by Lundstrδm et al.

(J. Biol. Chem. 267:9047-9052, 1992) and by a combination of 3) and 5) by Pigiet (WO 8906122) .

PDI, DsbA, TRX and ENG can be obtained by purification from 1) animal or 2) plant tissues, or from 3) microorganisms, or 4) by expression of recombinant DNA encoding plant, animal, human or microbial PDI, dsbA, TRX or ENG in microorganisms or other suitable hosts, followed by purification of PDI, dsbA, TRX or ENG from extracts or supernatants of said microorganisms. Preparation of TRX according to 1) was described by Luthman and

Holmgren (Biochem. 121:6628-6633, 1982) , according to 2) by

Wada and Buchanan (in "Thioredoxins, structure and function"

(Gadal, Ed.) Editions du Centre National de la Recherche

Scientifique) , according to 3) by Porque et al . (J. Biol. Chem. 245:2362-2379, 1970) and by Laurent et al. (J. Biol. Chem. 239:3436-3445) , and according to 4) by Krause et al. (J. Biol. Chem. 266:9494-9500) . PDI has been prepared according to 1) by Lambert and Freedman (Biochem J. 213:225-234, 1983) , according to 3) by Starnes et al. (US 4632905) and by Hammer et al . (US 4894340), and according to 4) by among others Yamauchi et al. (Biochem. Biophys. Res. Commun. 146:1485-1492, 1987) . Finally, an ENG was prepared by Lundstrδm et al . (J. Biol. Chem. 267:9047-9052, 1992) according to 4) .

Disulfide linkages in proteins are formed between cysteine residues and have the general function of stabilising the three dimensional structure of the proteins. They can be formed between cysteine residues of the same or different polypeptides.

Disulfide linkages are present in many types of proteins such as enzymes, structural proteins, etc. Enzymes are catalytic proteins such as proteases, amylases, etc., while structural proteins can be scleroproteins such as keratin, etc, protein material in hair, wool, skin, leather, hides, food, fodder, stains, and human tissue contain disulfide linkages. Treatment of some of these materials with PDI and TRX, and a redox partner has been described previously.

The use of TRX for waving, straightening, removing and soften¬ ing of human and animal hair was described by Pigiet et al . (EP 183506 and WO 8906122) . Pigiet (US 4771036) also describes the use of TRX for prevention and reversal of cataracts. Schreiber (DE 2141763 and DE 2141764) describes the use of protein disulfide transhydrogenase for changing the form of human hair. Pigiet (EP 225156) describes the use of TRX for refolding denatured proteins. Use of TRX to prevent metal catalysed oxidative damage in biological reactions is described by Pigiet et al. (EP 237189) .

Toyoshima et al. (EP 277563 and EP 293793) describe the use of PDI to catalyse renaturation of proteins having reduced disulfide linkages or unnatural oxidised disulfide linkages, in particular in connection with renaturation of recombinantly produced proteins. Brockway (EP 272781), and King and Brockway

(EP 276547) describe the use of PDI for reconfiguration of human hair, and for treatment of wool, respectively. Sulfhydryl oxidase for the treatment of Ultra-high temperature sterilized milk is described in US 4894340, US 4632905, US 4081328 and US 4053644. Schreiber (DE 2141763 and DE 2141764) describes the use of protein disulfide transhydrogenase for changing the form of human hair.

ABBREVIATIONS

A Adenine G Guanine C Cytosine T Thymine (only in DNA) U Uracil (only in RNA)

In the Tables "deletions" are indicated by "-", e.g. "SI--AKA" indicating that for this protein it appears as if two deletions have occurred compared to the other proteins in the Tables.

MUTATIONS

In describing the various mutants produced or contemplated according to the invention, the following nomenclatures were adapted for ease of reference:

Original amino acid(s) position(s) substituted amino acid(s)

According to this the substitution of Glutamic acid for glycine in position 195 is designated as: Gly 195 Glu or G195E a deletion of glycine in the same position is : Gly 195 * or G195* and insertion of an additional amino acid residue such as lysine is: Gly 195 GlyLys or G195GK

Where a deletion is indicated in the Tables, or present in a protein not indicated in the Tables, an insertion in such a position is indicated as: * 36 Asp or *36D for insertion of an aspartic acid in position 36

Multiple mutations are separated by pluses, i.e. : Arg 170 Tyr + Gly 195 Glu or R170Y+G195E representing mutations in positions 170 and 195 substituting tyrosine and glutamic acid for arginine and glycine, respec¬ tively.

SUMMARY OF THE INVENTION

The present invention relates to novel protein disulfide redox agents, DNA sequences encoding said novel protein disulfide redox agents, DNA constructs comprising said DNA sequences operably linked to sequences capable of directing transcription and translation of said DNA sequence in a suitable host microorganism, and optionally also being operably linked to sequences encoding signals enabling secretion of said protein

disulfide redox agents encoded by said DNA sequence to the extracellular medium, microorganisms containing said DNA constructs, and a process for producing protein disulfide redox agents.

The invention also relates to novel compositions of matter with improved properties for a number of applications and the use of said compositions in said applications. Said compositions comprise (i) a protein disulfide redox agent optionally in combination with (ii) at least one redox partner, and optional¬ ly (iii) one or more other enzymes.

BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES

Figure 1 displays the plasmid map of pCaHj435 made from the E.Coli expression vector pHD 389 (Lopez - Otin el. al. , J. Biol. Chem., in press) comprising the dsbA gen sequence.

Figure 2 displays the plasmid map of pPL1759 (Hansen. C. , Thesis, The Technical University of Denmark, 1992) .

Figure 3 displays the plasmid map of pJA146 made from the pPL1759 plasmid containing the putative mature dsbA encoding region (J.C.A. Bardwell et al. , Cell, 67, p. 581-589, 1991) .

Table 1 shows an alignment of published eukaryotic PDI amino acid sequences: Bovine (Bos taurus) (Yamauchi et al . , Biochem. Biophys. Res. Commun. 146:1485-1492, 1987), chicken (Gallus gallus) (Parkkonen et al . , Biochem. J. 256:1005-1011, 1988), human (Homo sapiens) (Rapilajaniemi et al . EMBO J. 6:643-649, 1987), mouse (Mus musculus) (Gong, et al . , Nucleic Acids Res. 16:1203, 1988) , rabbit (Oryctolagus cuniculus) (Fliegel et al. , J. Biol. Chem. 265:15496-15502, 1990) , rat (Rattus norvegicus) (Edman et al . , Nature 317:267-270, 1985) , and yeast (Saccharo- myces cerevisiae) (Tachikawa et al . , J. Biochem. 110:306-313) .

Table 2 shows an alignment of PDI amino acid sequences: Alfalfa (Medicago sativa) (Shorrosh and Dixon, Plant. Mol. Bio. 19:319-

321, 1992) , A. oryzae, yeast (Saccharomyces cerevisiae) (Tachi- kawa et al . , J. Biochem. 110:306-313) , bovine (Bos taurus) (Yamauchi et al . , Biochem. Biophys. Res. Commun. 146:1485-1492, 1987), rat (Rattus norvegicus) (Ed an et al . , Nature 317:267- 270, 1985), and mouse (Mus musculus) (Gong, et al . , Nucleic Acids Res. 16:1203, 1988) .

DETAILED DESCRIPTION OF THE INVENTION The novel protein disulfide redox agents of the invention comprises agents of the type ENG indicated above, wherein one or more of the sequences

has been changed to the following definition:

R ¹ and R ² each independently are different amino acid sequences identical to or different from the corresponding sequences in their parent molecule, X and Y each independently are any of the 20 other naturally occurring amino acids different from the ones in their parent molecule with the exception of TRX from E. coli , wherein His has been substituted for Pro as Y (E. coll P34H) , and DsbA from E. coli .

R ¹ and R ² are preferably of a length between 5 and 500 amino acid residues.

X is chosen among Ala, Val, Leu, He, Pro, Phe, Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asn, Gin, Asp, Glu, Lys, Arg and His, and Y is chosen among Ala, Val, Leu, He, Pro, Phe, Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asn, Gin, Asp, Glu, Lys, Arg and His.

In preferred embodiments X is chosen among Glu, Ala, Ser, Asp, Pro, Val and Y is chosen among Arg, Lys, Asn, Gin or Tyr.

In other preferred embodiments X is Gly, Val or Ala, while Y is any of the 20 other naturally occurring amino acids different

from the ones in their parent molecule, preferably Arg, Lys, Asn, Gin, Tyr or His.

In other preferred embodiments Y is either His, Pro or Tyr, while X is any of the 20 other naturally occurring amino acids different from the ones in their parent molecule, preferably Glu, Ala, Ser, or Asp.

With the exception of TRX from E. coll , wherein His has been substituted for Pro as Y (E. coli P34H) and dsbA from E. coli .

It is contemplated that not all sequences (A) in a specific variant or mutein have been changed in the same way, but it is preferred that all sequences (A) in a specific variant or mutein have been changed in the same way.

In yet further embodiments it is contemplated that the parent molecule is divided or truncated so as to provide for a smaller number of sequences (A) in each peptide, e.g. one PDI subunit with 2 CA) sequences or a split PDI, dsbA or TRX unit with only one (A) sequence; or wherein R ¹ and R ² different from those of the parent molecule. Naturally R and R ² may be made both shorter or longer.

The novel protein disulfide redox agents of the invention can be obtained through the methods indicated below.

As indicated the invention also relates to DNA sequences coding for the variants of the invention.

Many methods for introducing mutations into genes are well known in the art. After a brief discussion of cloning protein disulfide redox agent genes, methods for generating mutations in both random sites, and specific sites, within the protein disulfide redox agent gene will be discussed.

Cloning a protein disulfide redox agent gene

The DNA sequence of the DNA construct of the invention may be isolated by well-known methods. Thus, the DNA sequence may, for instance, be isolated by establishing a cDNA or genomic library from an organism expected to harbour the sequence, and screen- ing for positive clones by conventional procedures. Examples of such procedures are hybridization to oligonucleotide probes synthesized on the basis of any of the full amino acid sequences shown in Tables 1 and 2, or a subsequence thereof in accordance with standard techniques (cf. Sambrook et al . , 1989) , and/or selection for clones expressing a protein disulfide redox agent activity as defined above, and/or selection for clones producing a protein which is reactive with an antibody raised against the protein disulfide redox agent comprising any of the amino acid sequences shown in Tables 1 and 2.

A preferred method of isolating a DNA construct of the inven¬ tion from a cDNA or genomic library is by use of polymerase chain reaction (PCR) using degenerate oligonucleotide probes prepared on the basis of the amino acid sequence of the parent protein disulfide redox agent of the invention. For instance, the PCR may be carried out using the techniques described in US Patent No. 4,683,202 or by R.K. Saiki et al. (1988) .

Alternatively, the DNA sequence of the DNA construct of the invention may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers (1981) , or the method described by Matthes et al.

(1984) . According to the phosphoamidite method, oligonucleoti- des are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.

Finally, the DNA construct may be of mixed genomic and synthe¬ tic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) , the fragments corresponding to various parts of the entire recombinant DNA molecule, in accordance with standard techniques.

Generation of random mutations in the protein disulfide redox agent gene

Once the protein disulfide redox agent gene has been cloned into a suitable vector, such as a plasmid, several methods can be used to introduce random mutations into the gene.

One method would be to incorporate the cloned protein disulfide redox agent gene, as part of a retrievable vector, into a mutator strain of Eschericia coli .

Another method would involve generating a single stranded form of the protein disulfide redox agent gene, and then annealing the fragment of DNA containing the protein disulfide redox agent gene with another DNA fragment such that a portion of the protein disulfide redox agent gene remained single stranded. This discrete, single stranded region could then be exposed to any of a number of mutagenizing agents, including, but not limited to, sodium bisulfite, hydroxylamine, nitrous acid, formic acid, or hydralazine. A specific example of this method for generating random mutations is described by Shortle and Nathans (1978, Proc. Natl. Acad. Sci. U.S.A., 5: 2170-2174). According to the Shortle and Nathans method, the plasmid bearing the protein disulfide redox agent gene would be nicked by a restriction enzyme that cleaves within the gene. This nick would be widened into a gap using the exonuclease action of DNA polymerase I. The resulting single-stranded gap could then be mutagenized using any one of the above mentioned mutagenizing agents.

Generation of site directed mutations in the protein disulfide redox agent gene

Once the protein disulfide redox agent gene has been cloned, and desirable sites for mutation identified, these mutations can be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligonucleotide synthesis. In a preferred method, a single stranded gap of DNA, bridging the protein disulfide redox agent

gene, is created in a vector bearing the protein disulfide redox agent gene. Then the synthetic nucleotide, bearing the desired mutation, is annealed to a homologous portion of the single-stranded DNA. The remaining gap is then filled in by DNA polymerase I (Klenow fragment) and the construct is ligated using T4 ligase. A specific example of this method is described in Morinaga et al. , (1984, Biotechnology 2:646-639). According to Morinaga et al. , a fragment within the gene is removed using restriction endonuclease. The vector/gene, now containing a gap, is then denatured and hybridized to a vector/gene which, instead of containing a gap, has been cleaved with another restriction endonuclease at a site outside the area involved in the gap. A single-stranded region of the gene is then available for hybridization with mutated oligonucleotides, the remaining gap is filled in by the Klenow fragment of DNA polymerase I, the insertions are ligated with T4 DNA ligase, and, after one cycle of replication, a double-stranded plasmid bearing the desired mutation is produced. The Morinaga method obviates the additional manipulation of constructing new restriction sites, and therefore facilitates the generation of mutations at multi¬ ple sites.

Expression of protein disulfide redox agent mutants According to the invention, a mutated protein disulfide redox agent gene produced by methods described above, or any alterna¬ tive methods known in the art, can be expressed, in enzyme form, using an expression vector. An expression vector gen¬ erally falls under the definition of a cloning vector, since an expression vector usually includes the components of a typical cloning vector, namely, an element that permits autonomous replication of the vector in a microorganism independent of the genome of the microorganism, and one or more phenotypic markers for selection purposes. An expression vector includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes. To permit the secretion of the expressed protein, nucleotides encoding a "signal sequence" may be inserted prior to the coding sequence of the gene. For ex-

pression under the direction of control sequences, a target gene to be treated according to the invention is operably linked to the control sequences in the proper reading frame. Promoter sequences that can be incorporated into plasmid vec- tors, and which can support the transcription of the mutant protein disulfide redox agent gene, include but are not limited to the prokaryotic β-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl . Acad. Sci. U.S.A. 25:3727-3731) and the tac promoter (DeBoer, et al. , 1983, Proc. Natl. Acad. Sci. U.S.A. 8 _. 0:21-25) . Further references can also be found in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94.

The expression vector carrying the DNA construct of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be intro¬ duced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid, a bacteriophage or an extrachromo¬ somal element, minichromosome or an artificial chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome (s) into which it has been integrated.

In the vector, the DNA sequence should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell . Examples of suitable promoters for directing the transcription of the DNA construct of the invention, especially in a bacterial host, are the promoter of the lac operon of E. coli , the Streptomyces coelicolor agarase gene dagA promoters, the promoters of the Bacillus licheniformis α-amylase gene (amyL) , the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM) ,

the promoters of the Bacillus Amyloli<juefaciens α-amylase (a yQ) , the promoters of the Bacillus subtilis xylA and xylB genes etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding A . oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A . niger neutral α-amylase, A. niger acid stable α-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A . oryzae alkaline protease, A . oryzae triose phosphate isomerase or A. nidulans acetamidase.

The expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, poly¬ adenylation sequences operably connected to the DNA sequence encoding the protein disulfide redox agent of the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBHO, pE194, pAMBl, pHD 389 and pIJ702.

The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B . licheniformis , or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance. Examples of Aspergillus selection markers include amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance. Furthermore, the selection may be accomplished by co-transformation, e.g. as described in WO 91/17243.

While intracellular expression may be advantageous in some respects, e.g. when using certain bacteria as host cells, it is generally preferred that the expression is extracellular. The protein disulfide redox agents of the invention comprising a variant of any of the amino acid sequences shown in tables 1 or 2 may furthermore comprise a preregion permitting secretion of

the expressed protein disulfide isomerase into the culture medium. If desirable, this preregion may be native to the protein disulfide isomerase of the invention or substituted with a different preregion or signal sequence, conveniently accomplished by substitution of the DNA sequences encoding the respective preregions.

The procedures used to ligate the DNA construct of the inven¬ tion, the promoter, terminator and other elements, respect- ively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al . (1989) ) .

The cell of the invention either comprising a DNA construct or an expression vector of the invention as defined above is advantageously used as a host cell in the recombinant produc¬ tion of a polypeptide of the invention. The cell may be transformed with the DNA construct of the invention, conveni- ently by integrating the DNA construct in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromo¬ some may be performed according to conventional methods, e.g. by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.

The cell of the invention may be a cell of a higher organism such as a mammal, an avian, an insect, or a plant cell, but is preferably a microbial cell, e.g. a bacterial or a fungal (in¬ cluding yeast) cell.

Examples of suitable bacteria are gram positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus , Bacillus alkalo- philus, Bacillus amyloli<juefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Ba-

cillus thuringiensis , or Streptomyces lividanε or Streptomyces murinus , or gram negative bacteria such as E. coli . The trans¬ formation of the bacteria may for instance be effected by protoplast transformation or by using competent cells in a manner known per se .

The yeast organism may favourably be selected from a species of Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae . The filamentous fungus may advantageously belong to a species of Aspergillus, e.g. Aspergillus oryzae or Aspergil - lus niger. Alternatively, a strain of a Fusarium species, e.g. F. oxysporum, can be used as a host cell. Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se . A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023. A suitable method of transforming Fusarium species is described by Malardier et al. , 1989.

According to the invention expression of the DNA construct comprising the recombinant DNA sequence or expression vector carrying the DNA construct may take place as heterologous expression in a host cell different from the cell from where the recombinant DNA was derived.

According to the invention expression of prokaryote recombinant DNA may take place heterologously in cell compartments.

In preferred embodiment according to the invention the recombi- nant DNA derived from a cell e.g. of the genus Escherichia can be expressed in an other cell e.g. of the genus Bacillus or Streptomyces .

In another preferred embodiment of the invention the recombi- nant DNA derived from a cell e.g. of the genus Aspergillus can be expressed in a cell e.g. of the genus Bacillus or Streptomyces .

In a yet further aspect, the present invention relates to a method of producing a recombinant protein disulfide redox agent of the invention, which method comprises cultivating a host cell as described above under conditions conducive to the production of the protein disulfide redox agent and recovering the protein disulfide redox agent from the cells and/or culture medium.

The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the protein disulfide redox agent of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection) .

The resulting protein disulfide redox agent may be recovered from the medium by conventional procedures including separating the cells from the medium by centrifugation or filtration, if necessary after disruption of the cells, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, followed by purifi¬ cation by a variety of chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or the like.

It is of course also possible to produce the protein disulfide redox agent of the invention by culturing the filamentous fungal natural host or parent organism of interest and recover¬ ing the protein disulfide isomerase from the culture broth in traditional ways.

The present invention also relates to compositions comprising the protein disulfide redox agents of the invention, including also the variant of TRX from E. coli , wherein His has been substituted for Pro as Y (E. coli P34H) .

The compositions may suitably contain 0.01-200 mg of enzyme protein per gram, preferably 0.01-20 mg of enzyme protein per gram, especially 0.01-2 mg of enzyme protein per gram, or

alternatively 0.02-0.2 mg of enzyme protein per gram, or 0.01- 0.2 mg of enzyme protein per gram.

In another prefered alternative the composistion contain 0.01- 0.5 mg of enzyme protein per gram or alternativly 0.2-0.5 mg of enzyme protein per gram.

The compositions of the invention usually also comprises (ii) a suitable redox partner.

The redox partner (ii) is generally an organic or inorganic reductant, and would often be selected from the organic reductants, such as from the group comprising glutathione, L- cysteine, dithiothreitol, 2-mercaptoethanol, thioglycolic acid, L-cysteine ethylester, -mercaptoethylamine, mercaptosuccinic acid, jβ-mercaptopropionic acid, dimercapto adipic acid, thiomalic acid, thioglycolamides, glycol thioglycolate, glycerol thioglycolate, thiolactic acid and salts thereof.

Among inorganic reductants sulfite and bisulfite compounds will often be preferred.

Under this aspect the invention is not meant to comprise compositions comprising only naturally occurring TRX or PDI in combination with a redox partner. All types of ENG are nat¬ urally encompassed by the present invention also under this aspect.

Furthermore the compositions of the invention may optionally comprise (iii) another enzyme, where said other enzyme prefer¬ ably is selected among proteases, lipases, amylases, transglu- taminases, or another protein disulfide redox agent

Under this aspect the invention is meant to comprise composi- tions comprising all types of protein disulfide redox agents including naturally occurring TRX or PDI either without or in combination with a redox partner. All types of ENG are nat-

urally encompassed by the present invention also under this aspect.

The compositions of the invention may contain other ingredients known in the art as e.g. excipients, stabilizers, fillers, detergents, etc.

The compositions of the invention may be formulated in any convenient form, e.g. as a powder, paste, liquid or in granular form. The enzyme may be stabilized in a liquid by inclusion of enzyme stabilizers. Usually, the pH of a solution of the composition of the invention will be 5-10 and in some instances 7.0-8.5. Other enzymes such as proteases, cellulases, oxidases, peroxidases, amylases or lipases may be included in the compositions of the invention, either separately or in a com¬ bined additive.

The compositions of the invention can be used for the treatment or degradation of scleroproteins, especially hair, skin and wool, treatment and cleaning of fabrics, as additives to deter¬ gents, thickening and gelation of food and fodder, strengthen¬ ing of gluten in bakery or pastry products, and as pharmaceuticals for the alleviation of eye sufferings.

The present invention is further illustrated in the following examples which should not, in any manner, be considered to limit the scope of the present invention.

MATERIALS AND METHODS

Strain:

E. coli WA803 (Maniatis et al . , 1982, Molecular cloning, a laboratory manual, Cold Spring Habor Laboratory, New York) B . Subtilis DN1885 (P. L. Joergensen et al . , Gene, 96, p. 37- 41, 1990)

JA146 : B . subtilis DN1885 harbouring the pJA146 plasmid CaHj435 : E. coli harbouring the pCaHj435 plasmid

Plasmids : pCaHj435: Figure 1, plasmid comprising the dsbA gene sequence in E. Coli expression vector pHD389. pJA146: Figure 3, plasmid comprising the putative dsbA encoding region (J.C.A. Bardwell et al. Cell, 67, p. 581-589, 1991) in B . subtilis expression vector pPL1759 . pPL1759: B . subtilis expression vector (Hansen C, 1992, Thesis, The Technical University of Denmark) , figure 2. pHD389: E. coli expression vector, (Lopez - Otin et al. , J. Biol. Chem., in press)

Materials: Perkin Elmer- cetus Amplitaq™ Taq polymerase

DNA sequencing kit Sequenase™ (United States Biochemicals)

Super Taq™ DNA polymerase/PCR buffer, HT Biotechnology Ltd.)

Terrific Broth medium (Maniatis et. al (1982) Supra)

Terrific Yeast medium (PCT/DK9O/00332) LB agar (Luria-Bertani medium/agar, CR. Harwood and S.M.

Cutting (Ed.) Molecular Biological Methods for Bacillus, 1990,

John Wiley & Sons Ltd.)

DEAS Sphadex A-50 column (Pharmacia Fine Chemicals AB)

Methods:

Trypsin inhibitor assay (Available from Novo Nordisk A/S)

N-terminal amino acid sequence analysis

N- erminal amino acid sequence analysis of recombinant dsbA was carried out following electroblotting using an Applied Biosystems 473A protein sequencer operated according to the manufacturers instructions.

EXAMPLES

Example 1

Construction of the dsbA expression plasmid for expression in E. coli

The E. coli dsbA gene sequence was collected from the GenBank database (accession number M77746) . Based on this sequence a PCR primer containing the restriction enzyme Cla I recognition sequence and 23 bases of the dsbA 5' coding sequence (primer 5513) and a PCR primer containing the restriction enzyme Sail recognition sequence and 23 bases complementary to the dsbA 3' coding sequence (primer 5512) were constructed.

5513 5' CCATCGATGAAAAAGATTTGGCTGGCGCT 3 ' 5512 5' CCGTCGACTTATTTTTTCTCGGACAGATATT 3 '

Total DNA was extracted from E. coli strain WA803 using standard procedures.

This DNA was used without further modification as template in a PCR reaction (20 cycles) using the primers 5513 and 5512 and the Perkin Elmer- cetus Amplitaq™ Taq polymerase following the manufacturer's instructions.

A PCR fragment corresponding to the size of the dsbA gene was recovered from an agarose gel and digested with the restriction enzymes Clal and Sail.

The E. coli expression vector pHD 389 was digested with the same enzymes, and the large vector fragment was ligated to the digested PCR fragment. The ligation mixture was used to transform E. coli strain WA803. After 24 hours of growth at 30°C using ampicillin selection a transformant was selected and subsequent DNA sequence analysis using the DNA sequencing kit Sequenase™ showed that a sequence identical to the published dsbA gene sequence was integrated between the lambda PR promoter and the fd terminator. This plasmid was termed pCaHj 435, and the E. coli strain harbouring the plasmid was termed CaHj 435. A plasmid map of pCaHj 435 is shown in figure 1.

Expression of dsbA in E. coli phage lambda

The dsbA gene is under control of the promoter PR from the E. coli phage lambda. PR is repressed by CI repressor also harboured by the plasmid pCaHj 435. However the CI repressor allele used in this plasmid is temperature sensitive being active at 30°C but inactive at 42°C. Thus the dsbA gene is repressed at 30°C but expressed at 42°C.

In order to express the dsbA gene the strain CaHj 435 was grown in shake flasks containing the medium Terrific Broth at 30°C and 200 rpm until OD600 reached 0.2. Then the shake flasks were transferred to 42°C (200 rpm) for 18 hours.

Recovery and purification of the dsbA gene product. 1 liter of cell suspension was chilled on ice and then the periplasmic fraction of the cells was isolated by osmotic shock:

The cells were isolated by centrifugation (2500 x g, 15 min.) and resuspended in 100 ml 20% (W/V) sucrose buffered with 10 mM Tris/HCl pH 7.0. EDTA were added to a final concentration of 15 mM. The cell suspension was incubated on ice for 15 min. and then the cells were collected by centrifugation (2500 x g, 15 min.) . The cells were resuspended in 70 ml of water by vigorous shaking and subsequently incubated on ice (10 min) . The suspension was centrifuged (2500 x g, 15 min.) and the supernatant containing the soluble periplasmic fraction was isolated. Tris/HCl pH 7.0 was added to a final concentration of 5 mM.

The dsbA gene product was then purified by DEAE anion exchange chromatography.

A column containing 20 ml of DEAE Sephadex A-50 purchased from Pharmacia Fine Chemicals AB was equilibrated with 10 mM Tris/HCl pH 7.0. The osmotic shock preparation was applied to the column, and then the column was washed with 200 ml 10 mM Tris/HCl pH 7.0. The dsbA gene product was eluted with 50 ml 50

mM NaCl, 10 mM Tris/HCl pH 7.0. SDS polyacrylamide gel electro¬ phoresis showed that more than 90% of the protein isolated corresponded to the size of the dsbA gene.

Using the trypsin inhibitor assay it was shown that the purified protein has disulphide isomerase activity.

Example 2

Construction of the dsbA expression plasmid for expression in

Bacillus .

The E. coli dsbA gene sequence was collected from the GenBank database (acession number Will AS ) . Based on the dsbA sequence from GenBank and pCaHj435 (the dsbA expression plasmid in E. coli (WA803)) a PCR primer containing the restriction enzyme Nsil recognition sequence and 27 bases of the dsbA 5'sequence encoding the putative N-terminal (J.C.A. Bardwell et al . Cell, 67, p. 581-589, 1991) of the mature DsbA protein (primer 5965) and a primer containing a restriction enzyme EcoRI recognition sequence and 20 bases complementary to the dsbA 3' sequence of pCaHj435 (primer 5966) was made:

5965 5' -CCTCATTATGCATCAGCGGCGGCGCAGTATGAAGATGGTAAACAG-3' 5966 5' -GCGAATTCGTCGACTTATTTTTTCTCGG-3'

A reisolated colony of WA803/pCaHj435 grown 18 hours at 30°C on LB agar plates containing 100 μg/ml ampicillin, 10 mM potasium phosphate pH 7,0 and 0,4% glucose was resuspended in 10 μl 1 x PCR buffer (Super Taq™ DNA polymerase) heated to 99°C for 5 minutes, spunned 20 000 x g for 2 min.

5 μl of this supernatant was used as template in a PCR reaction (20 cycles) using the primers 5965 and 5966 and Super Taq™ DNA polymerase following the manufators instructions.

A PCR fragment corresponding to the expected size of dsbA was recovered from an agarose gel and digested with the restriction enzymes EcoRI and Pstl.

The plasmid pPL1759, fig. 2, was digested with the restriction enzymes Pstl-EcoRI and the large vector fragment was ligated to the PCR fragment. Ligation mixture was transformed into Bacillus subtilis DN1885. Selection for transformants and reisolation of those was performed on LB medium containing 10 μg Kanamycin/ml, 10 mM potasium phosphate pH 7,0, and 0,4% glucose.

DNA analysis of the plasmids from those cells using a DNA sequencing Kit (Sequenase™) showed the expected sequence of the promotor- and signal peptide encoding regions of amyL (P.L. Joergensen et al. , Gene, 96, p. 37-41, 1990) fused to the above mentioned putative mature dsbA encoding region. This plasmid was termed pJA146 and a B . subtilis DN1885 strain haboring this plasmid was termed JA146. A plasmid map of pJAl46 is shown in fig. 3.

Expression of dsbA in Bacillus

Strain JA146 was grown for 18 hours in Terrific Yeast medium at

37°C with 10 μg/ml Kanamycin and 0.4% glucose in 20 ml M-tubes at 280 rpm. Cells were harvested at 15 000 x g for 10 minutes and the supernatant was analysed for DsbA protein. SDS-PAGE of the supernatant showed that a protein of the size of mature DsbA protein was secreted into the media. Using the trypsin inhibitor assay it was shown that the DsbA protein has disulphide isomerase activity.

The N-terminal amino acid sequence was analysed as described above. The N-terminal amino acid of DsbA determined was : Ala- Ala-Gln-Tyr-Glu-Asp-Gly-Lys-Gln-

Example 3

The effect of waving composition on hair

Testing of the P34H variant of Thioredoxin from E. coli for enzymatic waving of hair.

A tress of washed human Scandinavian hair (1 gram) was wetted with water and tightly wound on a curling roller. 1 ml of a solution with the following composition and a temperature of 30°C was applied to the tress: 4 mg/ml P34H Thioredoxin 50 mM Phosphate buffer pH 7.5 1 mM Reduced Gluthation (Sigma)

The tress was put in a plastic bag and incubated for 60 minutes at 30°C. Then the roller was removed and the hair was rinsed with water, dried with a cotton towel, combed and air dried.

Other tresses of hair was treated like above but without addition of the P34H Thioredoxin variant.

Treatment with the P34H variant of Thioredoxin gives a sig¬ nificant waving effect on the hair.

Example 4

Waving effect on hair treated with DsbA The following experiments compare the effect of waving on hair treated with DsbA from E. coli and bovine PDI.

Tresses of washed brown and fair human European hair (1 gram) was wetted with water and tightly wound on curling rollers. 1 ml of waving solution with the following composition and temperature of 30°C was applied to the tresses.

0.38 mg/ml DsbA

lOmM Tris buffer pH 7.0 with 50mM NaCl 1 ml and 10 mM Reduced Gluthation (Sigma)

Tresses were put in plastic bags and stored for 60 minutes at 30°C. The hair was rinsed with water, dried with cotton towel, the rollers was removed, the hair was air dried and combed.

The results of the experiments are displayed in table A.

TABLE A

T e enght of all tresses were measured b.efore (Li _efore ) ^and after (L _a treatment .

^L a _teι J ^L ι _>e f _ore i- ^{s a} measurement for the waving effect on the hair .

Example 5

Control of permanent waving

To control that the enzymatic waving was permanent and not only temporary the hair tresses were washed with a mild commercial shampoo.

After being treated, as described in example 4 and rinsed, the hair tresses were removed from the curling rollers and dried. Then control tresses and enzyme treated hair tresses were washed in either water or a mild shampoo, rinsed and air dried.

The results of the test are displayed in table B,

TABLE B

af er _r / ^before is a measurement for the waving effect on the hair.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: NOVO NORDISK A/S

(B) STREET: Novo Alle

(C) CITY: Bagsvaerd

(E) COUNTRY: Denmark

(F) POSTAL CODE (ZIP) : DK-2880

(G) TELEPHONE: +45 4444 8888 (H) TELEFAX: +45 4449 3256

(ii) TITLE OF INVENTION: A method of producing a protein disulfide redox agent

(iii) NUMBER OF SEQUENCES: 4

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.3OB (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Primer"

(iii) HYPOTHETICAL: YES

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

CCGTCGACTT ATTTTTTCTC GGACAGATAT T 31

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Primer"

(iii) HYPOTHETICAL: YES

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

CCATCGATGA AAAAGATTTG GCTGGCGCT 29

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Primer"

(iii) HYPOTHETICAL: YES

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

CCTCATTATG CATCAGCGGC GGCGCAGTAT GAAGATGGTA AACAG 45

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Primer"

(iii) HYPOTHETICAL: YES

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

GCGAATTCGT CGACTTATTT TTTCTCGG 28

Table l:

1 50

Pdi_MOUSe MLS RALLCLALA AARVGADALE EED VLVLKK SNFEEALAAH

Pdi_Rat MLS RALLCLALAW AARVGADALE EEDNVLVLKK SNFAEALAAH

Pdi_Bovin MLR RALLCLALTA LFRAGAGAPD EEDHVLVLHK GNFDEALAAH

Pdi_Human MLR RALLCLAVAA LVR..ADAPE EEDHVLVLRK SNFAEALAAH

Pdi_Rabit MLR RAVLCLALAV TA.GWAWAAΞ EEDNVLVLKS SNFAEELAAH

Pdi_Chic EPLE EEDGVLVLRA ANFEQALAAH

Pdi_Yeast MKFSAGAVLS WSSLLLASSV FAQQEAVAPΞ DSA.WKLAT DSFNEYIQSH

51 100

Pdi_ .Mouse KYLLVEFYAP WCGHCKALAP EYAKRAAKLK AEGSEIRLAK VDATEESDLA Pdi. JRat NYLLVEFYAP WCGHCKALAP EYAKAAAKLK AEGSEIRLAK VDATEESDLA Pdi. Bovin KYLLVEFYAP WCGHCKALAP EYAKAAGKLX AEGSEIRLAK VDATEESDLA Pdi_ Human KYLLVEFYAP WCGHCKALAP EYAKAAGKLK AEGSEIRLAK VDATEESDLA Pdi. Rabit KHLLVEFYAP WCGHCKALAP EYAKAAGKLK AEGSDIRLAK VDATEESDLA Pdi. Chick RHLLVEFYAP WCGHCKALAP EYAKAAAQLK AEGSEIRLAK VDATEΞAELA Pdi Yeast DLVLAEFFAP WCGHCKNMAP EYVKAAETL. .VEKNITLAQ IDCTENQDLC

101 150

Pdi_ Mouse QQYGVRGYPT IKFFKNGDTA SPKEYTAGRΞ ADDIVNWLKK RTGPAATTLS Pdi_ Rat QQYGVRGYPT IKFFKNGDTA SPKEYTAGRΞ ADDIVNWLKK RTGPAATTLS Pdi ^" .Bovin QQYGVRGYPT IKFFKNGDTA SPKEYTAGRΞ ADDIVNWLKK RTGPAASTLS Pdi. _Human QQYGVRGYPT IKFFRNGDTA SPKEYTAGRΞ ADDIVNWLKK RTGPAATTLR Pdi_ _Ra i QQYGVRGYPT IKFFKNGDTA SPKEYTAGRΞ ADDIVNWLKK RTGPAATTLA Pdi. Chick QQFGVRGYPT IKFFRNGDKA APREYTAGRΞ ADDIVSWLKK RTGPAATTLT Pdi ^" Yeast MEHNIPGFPS LKIFKNSDVN NSIDYΞGPRT AEAIVQFMIK QSQPAVAWA

151 200

Pdi. Mouse DTAAAESLVD SSEVTVIGFF KDVESDSAKQ FLLAAEAIDD IPFGITSNSG Pdi] Rat DTAAAΞSLVD SSEVTVIGFF KDAGSDSAKQ FLLAAEAVDD IPFGITSNSD Pdi. Bovin DGAAAEALVE SSEVAVIGFF KDMESDΞAKQ FFLAAEVIDD IPFGITSNSD Pdi_ _Human DGAAAESLVE SSEVAVIGFF KDVESDSAKQ FLQAAEAIDD IPFGITSNSD Pdi. Rabit DSAAAESLVE SSEVAVIGFF KDVESDAAKQ FLLAAEATDD IPFGLTASSD Pdi] Chick DAAAAETLVD SSEVWIGFF KDVTSDAAKΞ FLLAAESVDD IPFGISSSAD Pdi Yeast DLPAYLANET FVTPVIVQSG KIDADFNATF YSMANKHFND YDFVSAENAD

201 250

Pdi_ Mouse VFSKYQLDKD GWLFKKFDE GR. . NNFEGΞ ITKEKLLD IKHNQLPLVI Pdi ^" .Rat VFSKYQLDKD GWLFKKFDE GR . .NNFEGΞ ITKEKLLD IKHNQLPLVI Pdi] Bovin VFSKYQLDKD GWLFKKFDE GR. . NNFEGE VTKEKLLD IKHNQLPLVI Pdi ^" .Human VFSKYQLDKD GWLFKKFDE GR. . NNFEGΞ VTKENLLD IKHNQLPLVI Pdi] Rabit VFSRYQVHQD GWLFKKFDE GR. . NNFEGE VTKEKLLD IKHNQLPLVI Pdi] Chick VFSKYQLSQD GWLFKKFDE GR. . NNFEGD LTKDNLLN.F IKSNQLPLVI Pdi ^" Yeast . .DDFKL . . . SIYLPSAMDE PWYNGKKAD IADADVFEKW LQVEALPYFG

251 300

Pdi_ Mouse EFTEQTAPKI FGGEIKTHIL LFLPKSVSDY DGKLSSFKRA AEGF..KGKI Pdi].Rat EFTEQTAPKI FGGEIKTHIL LFLPKSVSDY DGKLSNFKKA AEGF..KGKI Pdi]Bovin EFTEQTAPKI FGGEIKTHIL LFLPKSVSDY EGKLSNFKKA AESF..KGKI Pdi].Human EFTEQTAPKI FGGEIKTHIL LFLPKSVSDY DGKLSNFKTA AESF..KGKI Pdi ^" Rabi EFTEQTAPKI FGGEIKTHIL LFLPRSAADH DGKLSGFKQA AEGF..KGKI Pdi..Chick EFTEQTAPKI FGGEIKTHIL LFLPKSVSDY EGKLDNFKTA AGN ..KGKI Pdi Yeast EIDGSVFAQY VESGLPLGYL FY ND EEELEEYKPL FTELAKKNRG

table 1 (continued)

301 350

Pdi_MθUSΘ LFIFIDSDHT DNQRILEFFG LKKEECPAVR LITLEEEM TKY

Pdi_Rat LFIFIDSDHT DNQRILEFFG LKKEECPAVR LITLEEEM TKY

Pdi_Bovin LFIFIDSDHT DNQRILEFFG LKKEECPAVR LITLEEEM TKY

Pdi_Human LFIFIDSDHT DNQRILEFFG LKKEECPAVR LITLEEEM TKY

Pdi_Rabit LFIFIDSDHA DNQRILEFFG LKKEECPAVR LITLEEEM TKY

Pdi_Chick LFIFIDSDHS DNQRILEFFG LKKEECPAVR LITLEEEM TKY

Pdi Yeast LMNFVSIDAR KFGRHAGNLN M.KEQFPLFA IHDMTEDLKY GLPQLSEEAF

351 400

Pdi. Mouse KPESDELTAE K..ITEFCHR FLEGKIKPHL MSQEVPEDWD KQPVKVLVGA Pdi. Rat KPESDELTAE K..ITQFCHH FLEGKIKPHL MSQELPEDWD KQPVKVLVGK Pdi. Bovin KPESDELTAE ITΞFCHR FLEGKIKPHL MSQELPDDWD KQPVKVLVGK Pdi] Human KPESEELTAE ITEFCHR FLEGKIKPHL MSQERAGDWD KQPVKVPVGK Pdi. .Rabit KPESDELTAE ITEFCQR FLΞGKIKPHL MSQELPEDWD RQPVKVLVGK Pdi. Chick KPESDDLTAD IKEFCNK FLEGKIKPHL MSQDLPEDWD KQPVKVLVGK Pdi Yeast DELSDKIVLE SKAIΞSLVKD FLKGDASPIV KSQEIFENQD S.SVFQLVGK

401 450

Pdi_ Mouse NFEΞVAFDEK KNVFVΞFYAP WCGKCKQLAP IWDKLGETY. KDHENIIIAK Pdi. Rat NFEEVAFDEK KNVFVEFYAP WCGHCKQLAP IWDKLGETY. KDHENIVIAK Pdi] Bovin NFEΞVAFDΞK KNVFVΞFYAP WCGHCKQLAP IWDKLGETY. KDHΞNIVIAK Pdi_ Human NFΞDVAFDΞK KNVFVΞFYAP WCGHCKQLAP IWDKLGETY. KDHΞNIVIAK Pdi. .Rabit NFΞEVAFDEK KNVFVEFYAP WCGHCKQLAP IWDKLGETY. KEHQDIVIAK Pdi. .Chick NFEEVAFDΞN KNVFVΞFYAP WCGHCKQLAP IWDKLGETY. RDHΞNIVIAK Pdi ^" Yeast NHDEIVNDPK KDVLVLYYAP WCGHCKRLAP TYQELADTYA NATSDVLIAK

451 500

Pdi_Mouse MDSTANEVEA VKVHSFPTLK FFPASADRTV IDYNGERTLD GFKKFLESGG

Pdi_Rat MDSTANEVEA VKVHSFPTLK FFPASADRTV IDYNGΞRTLD GFKKFLΞSGG

Pdi_Bovin MDSTANEVEA VKVHSFPTLK FFPASADRTV IDYNGERTLD GFKKFLESGG

Pdi_Human MDSTANEVEA VKVHSFPTLK FFPASADRTV IDYNGΞRTLD GFKKFLΞSGG

Pdi_Rabit MDSTANEVEA VKVHSFPTLK FFPAGPGRTV IDYNGΞRTLD GFKKFLΞSGG

Pdi_Chick MDSTANEVEA VKIHSFPTLK FFPAGSGRNV IDYNGERTLE GFKKFLΞSGG

Pdi Yeast LDHTENDVRG WIEGYPTIV LYPGGKKSES WYQGSRSLD SLFDFIKΞNG

501 538

Pdi_ Mouse QDGAGDDEDL .DLΞΞ..ALΞ PDMΞΞ..DDD QKAVKDEL Pdi_ Rat QDGAGDNDDL .DLEΞ..ALΞ PDMΞE..DDD QKAVKDEL Pdi] Bovin QDGAGDDDDL EDLEΞ..AΞE PDLΞΞ..DDD QKAVKDΞL Pdi_ .Human QDGAGDDDDL ΞDLEΞ..AΞΞ PDMΞE..DDD QKAVKDEL Pdi_ .Rabit QDGAGDEDGL ΞDLΞΞ..AΞΞ PDLEE..DDD QKAVR EL Pdi] .Chick QDGAAADDDL ΞDLΞT..DΞΞ TDLΞΞGDDDΞ QKIQKDEL Pdi Yeast HFDVDGKALY ΞΞAQEKAAΞΞ ADADAELADE EDAIHDEL

Table 2

Alfalfa M-AKNVAIFG LLFSLLLLVP SQIFA--- --EES STDAKE

Oryzae MRTFAPWIL- --SLLGASA- --VAS -AADA TAEAPS

Yeast MKFSAGAVLS WSSLLLASS- --VFA-- QQEA VAPEDS

Bovine M-LRRA-LLC --LALTALF- --RAG-- --AGA PDEEDH

Rat M-LSRA-LLC --LALAWAA- --RVG ADA LEEEDN

Mouse MKLRKAWLLV LLLALTQLLA-AASAGDAQED TSDTENATEE EEΞEDDDDLΞ

-FVL- -DW- -AW- --VL- --VL-

VKEENGVWVL NDGNFDNFVA DKDTVLLEFY APWCGHCKQF APEYEKIAST - TLDNT SLTGD

- - KLATD -VLHKG VLKKS

LKDNDPPIAV AKIDATSASM LASKFDVSGY PTIKILKKGQ AVDYDGSRTQ NF HDTVKKHDFI WEFYAPWCG - TF ETFVKEHDLV LAEFFAPWCG

- - SF NΞYIQSHDLV LAEFFAPWCG NF DSALAAHKYL LVEFYAPWCG ^' -NF AEPAAKNYLL VEFY-APWCG

EEIVAKVREV SQPDWTPPPE VTLSLTKDNF DDWNNADII LVΞFYAPWCG

HCKKLAPΞYE KAASILSTHE PP LAKVDA NΞEHNKDLAS ENDVKGFPTI HCKALAPKYE QAATELKEKN IPL- -VKVDC TΞΞEA- -LCR DQGVEGYPTL HCKNMAPEYV KAAΞTLVΞKN ITL- -AQIDC TSNQD- -LCM EHNIPGFPSL HCKALAPEYA KAAGKLKAEG SEIRLAKVDA TΞΞSD- -LAQ QYGVRGYPTI HCKALAPΞYA KAAAKLKAEG SEIRLAKVDA TΞESD- -LAQ QYGVRGYPTI HCKKLAPΞYΞ KAAKΞLSKRS PPIPLAKVDA TΞQTD- -LAK RFDVSGYPTL

KIFRNGG-KN IQΞYKGPREA ΞGIVEYLKKQ SGPAS-TEIK SADDATAFVG KIFRGLDAVK P--YQGARQT EAIVSYMVKQ SLPAV-SPVT PENLEΞ-IKT KIFKNRDVNN SIDYEGPRTA EAIVQFMIKQ SQPAV-AWA DLPAYL-ANΞ KFFKNGDTAS PKEYTAGREA DDIVNWLKKR TGPAA-STLS DGAAAEALVE KFFKNGDTAS PKEYTAGREA DDIVNWLKKR TGPAA-TTLS DTAAAESLVD KIFRKG R PFDYNGPRΞK YGIVDYMIEQ SGPPSKEILT LKQVQEFLKD

DNKWIVGVF PKFSGEΞYDN FIALAEKLRS DYDFAHTLNA KHLPKGDSSV MDKIWIGYI ASDDQTANDI FTTFAESQRD NYLFAATSDA SI--AKAEGV TFVTPVIVQS GKIDADFNAT FYSMANKHFN DYDFVSAENA DD--DFKLSI SSEVAVIGFF KDMΞSDSAKQ FFLAAEVI-D DIPFGITSNS DV--FSKYQL SSEVTVIGFF KDAGSDSAKQ FLLAAEAV-D DIPFGITSNS DV--FSKYQL GDDWIIGLF QGDGDPAYLQ YQDAANNLRΞ DYKFHHTFSP EIAKFLKVSL

34

table 2 (continued)

SGPWRLFKP FDELFVDS-- -KDFNVEALE KFIEESSTPI VTVFNNEPSN KQPSIVLYKD FDEKKATYDG EIEQDALLSW VKTASTPLVG ELGPETYSGY YLPSAM--DE PWYNGKKAD IADADVFEKW LQVEALPYFG EIDGSVFAQY

DKDGWLFKK FD---EGR NNFEGEVTK EKLLDFIKHN QLPLVIEFTE

DKDGWLFKK FD---EGR NNFEGΞITK EKLLDFIKHN QLPLVIEFTE

GKLVLTHPEK FQSKYΞPRFH VMDVQGSTEA SAIKDYWKH ALPLVGHRKT

HPFWKFFNS PNAKAMLFIN FTTΞGAESFK TKYHEVAEQY KQQGV-SFLV ITAGIPLAYI FAETKEΞRΞQ FTΞΞFKFIAE KHKGSINIVT IDAKLYGAHA- VΞSGLPLGYL FYNDEEΞLEE YKPLFTELAK KNRGLMNFVS IDARKFGRHA QTAPKIFGGE IKTHILLFLP KSVSDYEGKL SNFKKAAESF KGKILFIFID QTAPKIFGGE IKTHILLFLP KSVSDYDGKL SNFKKAAEGF KGKILFIFID SNDAKRYSKR PLVWYYSVD FSFDYRAATQ FWRNKVLEVA KDFPEYTFAI

GDVESSQGAF QYFGLKEEQV PLI--IIQHN DGKKFFKPN- --LELDQLPT

GNLNLDPSKF PAFAIQDPEK NAKY -PYDQSKΞ- --VKAKDIGK

GNLNMK-EQF PLFAIHDMTE DLKYGLPQLS EEAFDELSDK IVLESKAIES SDHTDNQRIL EFFGLKKEEC PAVR-LITLE EEMTKYKPES DELTAEKITΞ SDHTDNQRIL EFFGLKKEEC PAVR-LITLE EEMTKYKPES DΞLTAΞKITQ ADEΞDYATΞV KDLGL-SΞSG ΞDVN-AAILD ΞSGKKFAMEP EEFDSDTLRΞ

WLKAYKDGKV EPFVKSΞPIP ETNN-EPVKV GQTLEDW FKSGKNVLIE FIQDVLDDKV EPSIKSEAIP ETQE-GPVTV WAHSYKDLV LDNEKDVLLE LVKDFLKGDA SPIVKSQEIF ENQD-SSVFQ LVGKNHDEIV NDPKKDVLVL FCHRFLΞGKI KPHLMSQELP DDWDKQPVKV LVGKNFEΞVA FDEKKNVFVE FCHHFLEGKI KPHLMSQELP EDWDKQPVKV LVGKNFEΞVA FDEKKNVFVE FVTAFKKGKL KPVIKSQPVP KN-NKGPVKV WGKTFDAIV MDPKKDVLIΞ

FYAPWCGHCK QLAPILDΞVA VSFQS-DADV VIAKLDATAN DIPTDTFDVQ FYAPWCGHCK ALAPKYΞELA SLYKD-IPΞV TIAKIDATAN DV--PD-SIT YYAPWCGHCK RLAPTYQΞLA DTYANATSDV LIAKLDHTEN DV--RGWIE FYAPWCGHCK QLAPIWDKLG ETYKD-HENI VIAKMDSTAN EV--EAVKVH FYAPWCGHCK QLAPIWDKLG ETYKD-HENI VIAKMDSTAN EV--EAVKVH FYAPWCGHCK QLΞPIYTSLG KKYKG-QKDL VIAKMDATAN DITNDQYKVΞ

GYPTLYFRSA SGK--LSQYD GGRT ΞDIIE FIE K NKDKTGAAHQ

GFPTIKLFAA GAKDSPVEYE GSRTVΞDLAN FVK E NGKHKVDALE

GYPTIVLYPG GKKSESWYQ GSRSLDSLFD FIK E NGHFDVDGKA

SFPTLKFFPA SADRTVIDYN GERTLDGFKK FLΞSGGQDGA GDDDDLEDLΞ SFPTLKFFPA SADRTVIDYN GΞRTLDGFKK FLESGRQDGA GDNDDLDLEE GFPTIYFAPS GDKKNPI K F -E GGNRDLEHLS

EVΞQPKAAAQ PE -" AΞQPKDΞL

VDPKKEQESG DATΞTRAASD ΞTETPAATSD DKSEHDEL LYΞEAQEKAA ΞEA ADAΞAE ADADAΞLADΞ EDAIHDΞL

EAEEPDLΞED DD QKAVKDEL

ALE-PDMΞΞD DD QKAVKDΞL

KF--ID-ΞHA TK RSRTKΞΞL