INTRODUCTION

Technical Field

The invention relates to transforming host cells, in particular yeast cells, for expression and secretion of peptides which inhibit ice crystal growth.

Background and prior art The success of a commercial process for the production of recombinant proteins requires a non-pathogenic organism amendable to large scale, high density fermenta¬ tion, e.g. Saccharomyces cerevisiae. Ideally, while the host organism should secrete relatively few proteins into the culture supernatant that could countermined or proteolytically degrade the product, it should have the capacity to secrete large quantities of correctly processed recombinant protein into the medium.

Antifreeze proteins (AFP\'s) from polar fish are characterized by their property of causing a noncolligative depression of freezing point, and beside this, these molecules inhibit the recrystallization of ice (Knight et al., 1984). Both types of activity are thought to result from the interaction of these cryoprotectants with the ice crystal. Within the winter flounder two different AFP\'s predominate, the HPLC-6 and the HPLC-8 (Fourney et al., 1984), both belonging to the type I class of AFP\'s (Davies et al., 1990), and they are the only one for which a x-ray crystal structure is known, and for which detailed structure /function relationships have been proposed (Yang et al, 1988,). In general, the 37 amino acids long molecule contains three 11 amino acids tandem repeats, with mainly alanine amino acids that favour a single α helix with a moderate amphiphilic character, having the majority of the hydrophillic amino acids along one face of the molecule, that are potential sites for hydrogen bonding to the oxygen atoms of the ice lattice. The AFP binds preferentially to the prism face, and thereby blocks the ice growth at the preferred growth site. The created dipole moment is thought to be the initial driving force for the specific recognition. The induced ordering of the water dipoles within the ice only allows further ice growth on the unordered basal plane, where

then AFP\'s bind again. This growth habit alteration causes the development of bipyramidal ice crystals, which are significantly smaller than normal water crystals (Davies et al, 1990).

Within E.coli fusions of synthetic or semisynthetic genes encoding antifreeze proteins and either truncated staphylococcal protein A gene (Mueller et al., 1991; Warren et al, 1990) or pro AFP\'s with β-galactosidase (Peters et al, 1989) were expressed intracellular . Different chimeric proteins containing different numbers of the 11 amino acid imperfect repeats were linked to the C-terminal end of protein A by a short amino acid spacer and tested for their recrystallization inhibition potential, whereby a protein with only two repeats lacked activity, while the inhibitory activity increased progressively for proteins containing three, four and five repeats, indicating that the number of homologous segments may determine the effectiveness of a single molecule in suppressing crystal growth, or may determine the molar number of molecules necessary to have an effect. Warren et al. also tested the expression of a slightly modified AFP-6 as an internal fusion with β-galactosidase in E.coli. Crude extract of the transformed cells displayed thereby a five-fold greater recrystallization inhibition than the negative control within the splat assay. For the foreign gene expression and secretion in yeast, the invertase (SUC2) signal sequence (Perlmann at al, 1982) has been used widely. Human α2-IFN (Chang et al, 1986) and human single chain urinary plasminogen activator (Melnick et al, 1990) have successfully secreted using this sequence, while the secretion of human α-1 anti- trypsin behind the SUC2 sequence caused problems (Moir et al, 1987). Attachment of a heterologous protein to the signal sequence often results in a change of at least the first amino acid C-terminal to the signal peptidase cleavage side. However, there appears to be flexibility in recognition of cleavage junction, since fusions of SUC2 with altered amino acid junctions, such as HSA or two forms of insulin, are still cleaved (Hitzeman et al, 1990). The second well described option for the expression and secretion of proteins and peptides within S. cerevisiae is the application of the pre-pro sequence of the α mating factor, one of the two oligopeptide hormones (pheromones) triggering sexual conjugation of the haploid cell types a and α (Betz et al, 1981). α-factor is a

13-amino acid peptide that is encoded by two unlinked structural genes, MFαl and MFα2. These genes encode precursors of 165 and 120 amino acids, respectively (Caplan et al, 1991). Both precursors include a signal, a pro region with three sites for N-glycosylation, and tandem α -factor repeats each of which is preceded by a spacer peptide. The 19 amino acid signal (pre) sequence is responsible for the initial targeting of the molecule to the secretory pathway, and is cut off by a signal peptidase, and after processing of the pro-part through proteolytic activities including Kex2 endoproteinase, Kexl carboxypeptidase and the dipeptidylpeptidase aminopeptidase in the golgi, the mature α mating factor is released into the surrounding medium.

The α leader segment mediated secretion from 5. cerevisiae is described in the literature for several different proteins and peptides, for example a consensus α-IFN, β-endorphin (Bitter et al, 1984), human-insulin like growth factor I expression (Steube et al, 1991), and human interleukin-6 expression (Guisez et al, 1991). The potential of α-MF leader sequence for high secretion, seems to he rather low (Rothstein et al, 1987), although more recent experiments indicate the specific relationship of leader sequence and structural protein to be more crucial for the success of such a process (Sleep et al, 1990). Moreover, recent results indicate the possibility that for some fusions the deletion of the complete pro-peptide sequence increases the secretion efficiency significantly (Achstetter et al, 1992).

SUMMARY OF THE INVENTION

According to one embodiment of the invention, a desired gene encoding a protein active in the inhibition of ice crystal growth is placed in frame as a fusion construct behind the α-galactosidase gene of Cyamopsis tetragonoloba. In order to be capable to regulate expression the new chimeric gene is placed under the control of the strong inducible GAL7 promoter. To obtain secretion of the α-galactosidase- AFP fusion protein into the surrounding medium the whole gene is placed behind the invertase signal sequence. According to another embodiment of the invention a gene encoding the desired anti¬ freeze peptide is directly expressed under the control of the inducible GAL7

promoter. To receive secretion the gene coding for the mature AFP is separated by the pre-pro sequence of the a mating factor from the promoter.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. a) Schematic presentation of plasmid pUR2650. b) schematic presentation of plasmid pUR2651.

Abbreviations used: a-gal: synthetic gene coding for α-galactosidase from Cyamopsis tetragonoloba. amp r.: β-lactamase gene, arrows indicate the direction of transcription, the size of the plasmid is mentioned in the Figure; the small box in front of the α- galactosidase gene represents the SUC2 signal sequence, afp: DNA coding for one copy of a synthetic AFP gene, the thicker line between α-galactosidase gene and AFP gene represents the IEGR sequence.

Figure 2. a) Schematic presentation of plasmid pUR2652, Xa-AFP indicates here the IEGR AFP coding sequence; LEU2d indicates the LEU2 gene with the truncated promoter. b) Schematic presentation of plasmid pUR2653.

Abbreviations used: GAL7-ISS: Gal7 promoter and SUC2 signal sequence;

2um = 2u = 2μm, the DNA coding for AFP is indicated as a solid box. LEU2 indicates the LEU2 gene with the intact promoter. T-pgk = T-PGK represents the

PGK terminating sequence. Figure 3. a) Schematic presentation of plasmids pUR2660 containing 2 copies of AFP separated by KREA. b) plasmid pUR2665, containing 3 copies of

AFP with the internal KREA spacers.

Figure 4. a) Schematic presentation of plasmid pUR2666, containing 4 copies of AFP and the internal KREA spacers and b) schematic presentation of plasmid pUR2667, containing 5 copies.

Abbreviations used are the same than in the description of Figure 1 and Figure 2.

Figure 5. Schematic presentation of plasmid pUR2674 containing the fusion between α-galactosidase and 5 copies of AFP, separated by the last 7 amino acids of the α-mating factor pro-sequence. Figure 6. a) Schematic presentation of plasmids pUR2676. b) pUR2677, the plasmids containing either 2 or 4 directly linked AFP gene cassettes behind the GAL7 promoter and the SUC2 signal sequence.

Figure 7. a) Schematic presentation of plasmids pUR2678. b) pUR2679; the plasmids containing either 2 or 4 directly linked AFP gene cassettes behind the GAL7 promoter and the pre-pro α -mating factor sequence.

Figure 8. a) SDS gel analysis. b) Western analysis of soluble cell fractions and culture fluids of yeast cells containing pUR2652.

Separation on a 8-18% PAA gradient gel. α-gal: purified α-galactosidase; c: soluble cell fraction; s: supernatant of cell cultures, b) The immune specific stain was done with anti-AFP antibody. The arrow indicates the band of the fusion construct.

Figure 9. a) SDS gel analysis. b) Western analysis of soluble cell fractions of 3 different transformants and 1 culture fluids of yeast cells containing pUR2653.

Separation on a 8-18% PAA gradient gel. α-gal: purified α-galactosidase; c: control = soluble cell fraction of host strain S. cerevisiae SU10; t: 3 different soluble cell fractions of transformants containing pUR2653. s: supernatant of cell culture.of transformant 1, 2 and 3 not shown, because identical, b) The immune specific stain was done with anti-AFP antibody, c: control = culture fluid of untransformed SU10 cultures. The arrow indicates the band of AFP.

Figure 10. Graphical presentation of the results of the thermal hysteresis assay.

The difference in °C between freeze- and melting point is given on the vertical axis. A: chemically synthesized purified AFP at a final concentration of 3%.

B: purified α-galactosidase at a final concentration of 3%.

C: An about 40% suspension of freeze dried culture fluid of yeast cultures containing pUR2652.

D: an about 40% suspension of freeze dried culture fluid of yeast cultures containing pUR2653. For further details see text.

Figure 11. Recrystallization assay of culture fluid from yeast cells containing pUR2652.

A: 30% sucrose, 1). before and 2) after incubation.

B: 3% chemically synthesized AFP in 30% sucrose 1) before and 2) after incubation. C: about 40% freeze dried culture medium from cells harbouring pUR2652, a) before and b) after incubation, magnification were identical in all cases.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The invention particularly relates to a process for producing peptides inhibiting ice crystal growth, so-called anti-freeze peptides (AFP), by expressing in a host organism a gene encoding an AFP or encoding a fusion protein comprising an AFP, which comprises

(i) forming a DNA construct comprising in the order given

(a) a strong, optionally inducible, promoter active in the host organism,

(b) an ATG start codon, that may be contained in an optionally present DNA signal sequence capable of secreting the protein produced by the host organism during expression of an AFP-encoding gene of (c) below, which signal sequence can be homologous or heterologous to the AFP-encoding gene and is in reading frame with the ATG start codon,

(c) at least one desired AFP-encoding gene in reading frame with the ATG start codon, and (d) a stop codon bound to the 3\' end of the AFP-encoding gene,

(ii) transforming a host organism with the DNA construct of (i) in such a way that the host organism can express at least the AFP-encoding gene, (iii) growing the transformed host organism under conditions whereby the expression of at least the AFP-encoding gene occurs or is induced, and (iv) optionally collecting the produced AFP or fusion protein, or peptide or protein obtained therefrom by further processing.

The "host organism" can be a plant or animal cell or a microorganism. Preferably a yeast, e.g. S. cerevisiae is used as the host organism. Transformation systems for these host organisms are well known to a skilled person and are described in the prior art (see above).

In case of a "fusion protein" good results were obtained with a process according to the invention, in which the DNA construct contains a fused gene comprising at least part of the α-galactosidase gene of Cyamopsis tetragonoloba and at least one AFP-en¬ coding gene with its 5\' end in-frame-fused to the 3\' end of the at least part of the α- galactosidase gene. The fused gene was preceded by the invertase signal sequence of S. cerevisiae.

The "promoter" can be an inducible GAL7 promoter or a constitutive GAPDH promoter, both of S. cerevisiae. The advantage of an inducible promoter is that first sufficient biomass can be formed of the host organism which can then be induced to express the desired gene. However, a constitutive promoter is preferable under conditions where it is advantageous to express the desired gene in all growth stages. Of course the gene should comprise or be preceded by an "ATG start codon" for starting proper translation of the mRNA.

If the gene or fused gene is to be secreted a DNA "signal sequence" can be used. Most signal sequences precede the structural gene and start with an ATG start codon. As DNA signal sequence can be used for example the invertase signal sequence of S. cerevisiae or the DNA pre-sequence of the α-mating factor of 5. cerevisiae. In the latter case the DNA construct can also comprise the pro-sequence of the α-mating factor of S. cerevisiae between the pre-sequence and the AFP-encoding gene, whereby the pre-sequence, the pro-sequence and the AFP-encoding gene are in the same reading frame. _

When it is desirable that the AFP remains in the cell of the host organism and secretion is less desirable, a signal sequence need not be present. Of course the AFP need then not be collected from the host organism. On the other hand, when the presence of the AFP is desirable, a signal sequence is preferred for obtaining efficient secretion. Thus the presence of a signal sequence is optional depending on the purpose of the AFP production. Any signal sequence present can be both homologous to the AFP, thus corresponding to the AFP in its natural occurrence, or can be heterologous to the AFP. The latter can have the advantage that it is better recognized by the host organism. For proper processing after secretion it can be advantageous when the connection between two or more genes comprises a DNA sequence encoding a "processing site" that can be used to split off at least one AFP from the remainder of the protein, either during or after secretion of the fusion protein or another precursor- AFP. It should be noted that in this specification the "at least one AFP-encoding gene" can comprise a tandem-repeat of one or more AFP-encoding genes, whereby optionally one or more AFP-encoding genes can be separated by a DNA sequence encoding a processing site.

The invention also relates to an AFP prepared by a process according to the invention and to the use of such AFP for inhibiting ice crystal growth, e.g. in ice cream and other food products, or other biological materials that can be frozen. Another embodiment of the invention relates to a yeast having improved freeze tolerance, containing and/or surrounded by AFP produced according to the invention, which can be used in producing frozen dough or other compositions that can be frozen.

The invention is illustrated by the following examples without being limited thereto. Mostly standard methods were used as described in Sambrook, J., Maniatis, T., Fritsch, E.F.; Molecular Cloning; Second edition; Cold Spring Harbour Laboratory Publ. (1989). If modifications were used, then they are described below.

Strains, plasmids and growth conditions

E. coli strain JM109 ( endAl, recAl, syrA96, thi, hsdR17, rk ^" , mk ⁺ relAl supE44, Yanish-Perron et al, 1985) was used for amplification of plasmids. Transformation of

JM109 was according to Chung et al, 1989. S. cerevisiae strain SU10 (matα, cir ⁺ ,

MEL ⁺ , leu2, his3, ura3) was transformed with the plasmids pUR2652 and pUR2653.

S. cerevisiae SU50 (mata, cir°, leu2, his4, canl; Erhart and Hollenberg, 1981) was used for transformation of the multicopy integration plasmid pUR6803. Transformation of the yeast strains was performed by the spheroplast method of

Beggs et al or by electroporation, mainly as described by Becker et al, 1991.

Transformants were recovered on selective YNB-plates (0.67% YNB, 2% glucose,

2% agar) supplemented with the essential amino acids ( histidine 20μg/ml, uracil

20μg/ml). The same liquid medium was used for pre-cultures, 2 times overnight at 30°C and diluted 1:10 in YP medium containing 5% galactose for induction of the

GAL7 promoter. Restriction enzymes and DNA modification enzymes were applied as recommended by the supplier.

Oligonucleotides were synthesized on an Applied Biosystems 380A DNA synthesizer and purified by standard procedures.

EXAMPLE 1: Construction of pUR2652

Plasmid pUR2652 was constructed to obtain a C-terminal fusion of AFP to α-galacto¬ sidase, a protein from which is known, that it is efficiently secreted into the medium, when fused to the invertase signal sequence (Verbakel, 1991). The invertase signal sequence directs proteins to the secretory pathway, and will be cut of by a signal peptidase. On the DNA level the whole gene is controlled by the inducible GAL7 promoter. For the expression of AFP-6 in S. cerevisiae a set of 8 oligonucleotides was synthesized (AFL 03, AFP 01, AFP 03, AFP 05, AFL 04, AFP 02, AFP 04, AFP 06), mainly comprising the DNA sequence of the mature AFP expressed in preferentially used S. cerevisiae codons (see SEQ.ID. No. 1 and 2).

_Γ AFL03 _τ AFP01

Styl CAAGGTCT ATT GAA GGT AGA GAT ACT GCT TCT GAT GCC GCC GCT GCC GCT

CAGA TAA CTT CCA TCT CTA TGA CGA AGA CTA CGG CGG CGA CGG CGA L AFL04 -L

_τ AFP03 _r

GCT TTG ACA GCT GCT AAT GCT AAA GCC GCT GCT GAA TTG ACA GCT GCC AAT CGA AAC TGT CGA CGA TTA CGA TTT CGG CGA CGA CTT AAC TGT CGA CGC TTA AFP02 -1 AFP04

AFP05 GCA GCC GCT GCT GCT GCA GCT ACT GCG AGA TAATA CGT CGG CGA CGA CGA CGT CGA TGA CGC TCT ATTAT CGA ffi-jdlll ^{J L} AFP06

The cassette further contained at the 5\' end codons for the in frame expression of the amino acid sequence IEGR, which is known to be the recognition and cleavage sequence for the blood factor Xa (Nagai et al, 1987). This construction allowed the later release the pure AFP from the fusion protein. For cloning purposes the nucleotides for the C-terminus of α-galactosidase from the restriction site Styl were also included. The 3\' end of the cassette included 2 stop codons, which partially overlap the H dlll restriction site. To facilitate the ligation of the assembled cassette behind the α-galactosidase gene, the Sαcl/H dlll fragment of pUR2740, comprising the 3\' end of the GAL7 promoter, the SUC2 signal sequence and the complete synthetic α-galactosidase gene (Verbakel, 1991) was subcloned into pTZ19R

(Pharmacia), thereby generating the construction intermediate pUR2650 (Figure la),

in which the Styl and Hz>ιdIII sites used for the integration of the ohgonucleotide cassette are unique.

For the assembly of the synthetic AFP gene, 50 pmol of each of the oligonucleotides, except AFL03 and AFP06, the ones at the 5\' overlapping end of the cassette, were dissolved in 12 μl water, incubated for 2 min. at 95°C, and directly placed on ice. After this denaturation step, the oligonucleotides were phosphorylated in a final volume of 20μl, containing 2.5mM ATP, 5 mM DTT and about 10 U of polynucleotid-kinase, for 40 min. at 37°C, followed by a 2 min denaturation at 95°C and placement on ice. lOμl of each phosphorylated ohgonucleotide was mixed with the corresponding DNA ohgonucleotide to obtain duplex formation. After 2 min 95°C denaturation, each duplex was slowly cooled down to 30°C. Again lOμl of all five duplex mixtures were pooled and incubated in a final volume of lOOμl, containing 50 mM Tris/ΗCl, pΗ 7.5, 8 mM MgC12, 8 mM DTT, and 40 μg/ml gelatine and 10 U of DNA ligase, for two hours at 20°C. The ligation mix was then precipitated, and redissolved in 30 μl of TE-buffer. 15 μl of the mixture were placed on a 2 % agarose gel, and the DNA band of the expected size was cut out of the gel and finally purified through the gene clean II procedure, as recommended by the supplier. The obtained DNA fragment was then ligated into the Styl/ Hindlϊl linearized vector pUR2650 and transformed into E.coli JM 109 by standard procedures, plasmid DNA of several transformants was isolated by the slightly modified alkaline-lysis mini-preparation method and analyzed by restriction analysis with several enzymes. This intermediate construct, containing the fusion cassette between α-galactosidase- and AFP-6 gene was named pUR2651 (see Figure lb). The new 1.3 kb Sacl/HindlU fragment was than reintroduced into the SαcI/Hndlll cut 2μ shuttle vector pUR2740 and transformed to E.coli. Several transformants were analyzed for the presence of the expected fragment by restriction analysis. From the presumably positive plasmids the correct assembly construction was confirmed by Sanger dideoxy sequencing of double stranded plasmids. This plasmid is designated pUR2652 (Figure 2a)

EXAMPLE 2 : Construction of pUR2653

By exchanging the DNA sequence coding for the α-mating factor against DNA coding for the anti freeze-peptide, we tried to obtain the mature AFP in the medium as a result of solely yeast borne proteolytic processing. For this purpose the six core- oligonucleotides (AFPOl, AFP03, AFP05, AFP02, AFP04 and AFP06), comprising the major part of the AFP coding sequence as well as the double stop codon and the Hindlϊl restriction site, were annealed to a new pair of oligonucleotides (AFLOl and AFL02), coding for an Eagl site, resp. an extra Ala at the N-terminus of AFP-6 and the first 4 amino acids (see SEQ. ID. NO: 3 and 4).

_| - AFLOl _τ AFPOl η

Eagl GGCC GAT ACT GCT TCT GAT GCC GCC GCT GCC GCT GCT TTG ACA GCT GCT CTA TGA CGA AGA CTA CGG CGG CGA CGG CGA CGA AAC TGT CGA CGA ^L AFL02 J L _r AFP03 _Γ

AAT GCT AAA GCC GCT GCT GAA TTG ACA GCT GCC AAT GCA GCC GCT GCT GCT

TTA CGA TTT CGG CGA CGA CTT AAC TGT CGA CGC TTA CGT CGG CGA CGA CGA

AFP02 J- AFP04 ^J AFP06

AFP05

GCA GCT ACT GCG AGA TAAT A

CGT CGA TGA CGC TCT ATTA TTCGA Hindlll

J

This synthetic genes simply generates an in frame transition between the coding part of AFP and the Eαgl site at the end of the prepro-sequence in vector PSY16, a 2μm derived shuttle vector for leucine complementation, where the C-terminal end of the natural prepro α mating factor sequence, was changed in such a way, that ligation of DNA via Eagl fuses the introduced coding part to the prepro-sequence. The DNA oligonucleotides were assembled into a double strand DNA fragment and purified as described in example 1 and the obtained 120 nucleotides long fragment was directly ligated into the Eagl/Hindlll cut PSY16 vector. Some of the obtained transformants were grown up, the plasmid DNA isolated, and the construction controlled by restriction analysis and verified by dideoxy sequencing of the double stranded plasmids; This plasmid was designated pUR2653 (Figure 2b).

EXAMPLE 3: Construction of pUR2660, pUR2665, pUR2666 and pUR2667

To obtain a vector with more than one gene coding for AFP-6 we exchanged the ohgonucleotide pair AFP05/AFP06 against a new set of oligonucleotides (AFL05/AFL06), comprising the C-terminus of the AFP and the four amino acid extension Lys, Arg, Glu, Ala, KREA, the proteolytic core part of the preproα mating factor spacer sequence, necessary to obtain mature N- and C terminal ends of the mating factor. The 3\'-end oligonucleotides were designed in such a way, the they could be directly ligated into the Eagl site of pUR2653, thereby leaving only the new 5\' end an intact Eagl restriction site, which then could be used for further additions of AFP cassettes.

_r AFLOl _τ AFPOl

Eagl GGCC GAT ACT GCT TCT GAT GCC GCC GCT GCC GCT GCT TTG ACA GCT GCT CTA TGA CGA AGA CTA CGG CGG CGA CGG CGA CGA AAC TGT CGA CGA AFL02 -L _r AFP03 -, _Γ

AAT GCT AAA GCC GCT GCT GAA TTG ACA GCT GCC AAT GCA GCC GCT GCT GCT TTA CGA TTT CGG CGA CGA CTT AAC TGT CGA CGC TTA CGT CGG CGA CGA CGA AFP02 J- AFP04 AFL06

AFL05 GCA GCT ACT GCT AGA AAG AGA GA CGT CGA TGA CGA TCT TTC TCT CTCCGG [iϊagl]

For that purpose the vector pUR2553 was linearized with Eαgl, dephosphorylated by standard procedures, and the totally phosphorylated AFP cassette was ligated into the vector, transformed into E.coli JM109, several transformants analyzed as described and the correct assembly verified by DNA sequencing; the new recombinant plasmid was designed pUR2660 (Figure 3a). Subsequent reassembly and reintroduction of the same ohgonucleotide cassette into the unique Eαgl site in the transition part between signal sequence and AFP coding part resulting in plasmids containing the DNA coding for 2 AFP\'s (pUR2660), 3 AFP\'s (pUR2665), 4 AFP\'s (pUR2666) and 5 AFP\'s (pUR2667). In each of these constructs every coding part is separated by the DNA coding for the KREA sequence (Figure 3a, 3b, 4a and 4b).

EXAMPLE 4: Construction of pUR2661

Trypsin is known to digest polypeptides carboxyterminal to amino acids Lys and Arg. Since amino acid sequence analysis revealed that Arg is the last amino acid of the mature AFP, digestion with this protease in principle allows the separation of AFP monomers from a given poly AFP peptide. But to leave the AFP in such a proteolytic digestion completely intact, the unique Lys has to be changed in the coding sequence. Structural analysis revealed, that this Lys is located on the more hydrophobic site of the molecule (Yang et al, 1988, Davies et al, 1990), a change into a neutral charged Ala further increases the amphiphilic character of the α helical structure. In extension of these options allowing the in vivo processing and maturation of poly-AFP precursor molecules, constructs were made, in which the unique Lys at amino acid position 18 within the AFP is exchanged against an Ala, the main amino acid of the AFP-6. For the strongly related AFP-8 from winter flounder it was suggested, that amino acids Lys22 and Asp26 form a salt bridge, which is functional in stabilizing the α-helical configuration (Chakrabartty et al, 1989). In a very recently published article, Mueller et al, 1991, described a possible synergy between the C-terminal Arg and the salt bridge in AFP-8, whereby salt bridge and carboxyl Arg together do not affect the recrystallization inhibition activity, whereas either feature alone tends to reduce it.

Such a salt bridge seems also to be present in AFP-6, between lyslδ and Glu22, which might have a similar stabilizing effect. The exchange of this Lys therefore not only allows the trypsin digestion of the direct fusion polypeptide, but also gives an idee about the contribution of this salt bridge in AFP-6 to the crystallization inhibition activity.

_Γ AFLOl -, _r AFPOl

Eagl GGCC GAT ACT GCT TCT GAT GCC GCC GCT GCC GCT GCT TTG ACA GCT GCT CTA TGA CGA AGA CTA CGG CGG CGA CGG CGA CGA AAC TGT CGA CGA L AFL02 ^J

_j - AFP13 _r

AAT GCT GCT GCC GCT GCT GAA TTG ACA GCT GCC AAT GCA GCC GCT GCC GCC TTA CGA CGA CGG CGA CGA CTT AAC TGT CGA CGC TTA CGT CGG CGA CGG CGG AFP12 ^{J L} AFP04 AFL08

AFL07

GGC TGC TAC TGC AAG AGA TAC TGC TAG C CCG ACG ATG ACG TTC TCT ATG ACG ATC GCT ACGGCGGCGA

From the already described set of "core" oligonucleotides AFLOl, AFL02, AFPOl, AFP04 were used in combination with the newly synthesized oligonucleotides AFP12 and AFP13, which form a duplex displaying the central part of the AFP, with the mentioned Lys to Ala exchange at position 18. The second new pair of oligonucleo¬ tides AFL08 and AFL07 just served for the direct linkage of the coding sequences. The ohgonucleotide pairs AFL01/AFL02, AFL07/AFL08, AFP05/AFP06, AFP01- /AFP12 were therefore used at a concentration of about 25 pmol, while the common structural parts of both copies, displayed by AFP01/AFP12 and AFP13/AFP04, were used at a final concentration of 50 pmol (Figure 6).The assembly was done as described, generating a Eαgl/Hindlll fragment with two directly linked APF coding parts, where in both genes the Lys codon was exchanged against Ala. This plasmid was named pUR2661 and is identical to pUR2660, with the exception of the 12 nucleotides coding for the KREA amino acid sequence and the mentioned Lys to Ala exchange.

EXAMPLE 5: Construction of pUR2674

Another possibility for increased AFP peptide expression in yeast is the repeated fusion of AFP-6 behind the carrier protein α-galactosidase, separated by internal spacers originating from the intervening sequences of the α-mating factor precursor molecule. To obtain a DNA template coding for 5 repeats of AFP-6 separated by KREA PCR technique was applied. Here, plasmid pUR2657 served as template during the reaction. The primers were designed in such a way, that an in frame

fusion between the end of the α-galactosidase gene at the Styl site at the 3\' end of the gene, -already used for the construction of pUR2652-, and the AFP coding part was insured. Both parts were again separated by the last 6 amino acids of the α-mating factor pro sequence and an additional extra alanine at the N terminus of the first AFP, which is a result of the introduction of the Eagl site for cloning purposes.

5\' CCC CCA AGG TCT AAA AGA GAG GCT GAA GCG GCC pAFCOl stx in III in in III III III pUR2667 3\' TTT TCT CTC CGA CTT CGC CGG 5\'

5\' AAA AGA GAG GCT GAA GCG GCC GAT ACT 3* K R E A E A A D T

The corresponding primer at the 3\' end of the gene was synthesized in such a way, that a unique hybridization on the template was ensured, thereby leaving the Hindlll site behind the stop codon intact.

Hindlll primer AFC02 CGC TCT ATT ATT CGAATAATA 5\'

III III III III 111111111 pUR2667 5\' AC5 ^" GCG AGA TAA TAA GCTTATTATCTCGC 3" 3\' TGA CGC TCT ATT ATT CG ATAAT GAGCG 5" T A R

PCR amplification of the template was carried out in a Perkin Elmer Cetus DNA Thermal Cycler. The reaction was carried out in 100 μl 10 mM Tris, HC1, pH 8.3, 50 mM KC1, 1.5 mM MgCl ₂ , 0.001% gelatine, with 0.2 mM of each dNTP, 100 pmol of the DNA oligonucleotides KLM06 and KLM09, about 0.1 μg of EcoRI digested DNA and 1 U of Amplitaq polymerase. Incubation parameters were set as follows: 32 cycles/ 1 min 95 °C/ 2 min 50 °C/ 2,5 min 72 °C. About 20μl of the reaction volume was placed on a 1% agarose gel, the DNA fragment of about 650 bp isolated and purified with the Genecleanll kit (Bio 101 Inc.) according to the manufacturers protocol. The fragment was subsequently digested with Styl and Hindlll and ligated into the Styl/Hwdlll digested plasmid pUR2650. After transformation into E.coli JM109 the new plasmid was isolated and digested with SαcI/Ht ^\' /idlll. The about

1.85 Kbp large fragment comprising the DNA coding for the SUC2 signal sequence and the α-galactosidase (5x)AFP fusion was subsequently ligated into the vector pUR2741, whereby plasmid pUR2741 is a derivative of pUR2740 (Verbakel, 1991), where the second Eagl restriction site in the already inactive Tet resistance gene was deleted through Nrul/Sall digestion.

One of the positive clones, designated pUR2674 (Figure 5), was further charac¬ terized, the DNA isolated and purified according to the Qiagen protocol and subsequently characterized by DNA sequencing. DNA sequencing was mainly performed as described by Sanger, and Hsiao, here with the Sequenase version 2.0 kit from United States Biochemical Company, according to the protocol with T7 DNA polymerase (Amersham International pic) and [ ³⁵ S]dATPαS (Amersham International pic: 370 MBq/ml; 22 TBq/mmol).

EXAMPLE 6: Construction of pUR2676, pUR2677, pUR2678 and pUR2679 Another option for the tandem expression of AFP\'s in yeast is the direct fusion of AFP coding part to each other, without altering the coding sequence. Here again the unique presence of Arg at the C-terminal position mature AFP-6 is Arg, allows a post-fermentative digestion of the fusion product into single monomers. This can be obtained through either the addition of carboxypeptidase Arg directly to the yeast culture supernatant or the partially or completely purified culture medium.

Therefore again PCR technique was applied. Through the use of 2 corresponding primers, complementary to both ends of the AFP-6 coding sequence, DNA frag¬ ments were generated, encoding only structural information of AFP. For the direct linkage of the PCR fragment to the first copy already present in pUR2653, the Pstl site present in the 3\'-part of the AFP coding sequence was used. Therefore, the 5\' PCR ohgonucleotide AFPPRIJVA also enclosed the DNA sequence coding for C-terminal part of AFP from the -Pstl site on. The corresponding 3\' PCR ohgo¬ nucleotide AFPR2JVA contained a sequence extension at the 5\' end comprising a Stal starting with the last codon of the AFP gene, to ensure the direct in frame blunt end linkage between different AFP copies (see SEQ. ID. NO: 15 - 20).

PCR primer AFPPRIJVA:

pUR2653 5\' GAT ACT GCT TGT GAT GCC GCC GCT GCC

3\' CTA TGA CGA ACA CTA CGG CGG CGA CGG Psti HI || || in ||| ||| III III III

5\' CCCCC CTGCA GCT ACA GCT AGG GAT ACC GCC TGT GAT GCC GCC GCT GCC A T A R D T A S D A A A A

PCR primer AFPR2 VA: StuI -fiΛdlll

CGA CGA CGA CGC CGA TGA CGC TCC GGA ATTCGAACGTAC 5\'

III III III II III III III II

5\' GCC GCT GCT GCT GCA GCT ACT GCG AGA 3 \' CGG CGA CGA CGA CGT CGA TGA CGC TCT A A A A A T A R

Primer AFPR2JVA also inactivated the -Pstl site present in the template, without altering the amino acid sequence. This allowed the Pstl/ Hindlll digestion of the PCR product, which was generated and purified as described earlier. To facilitate further cloning procedures, the vector pUR2650 was digested with Eagl/ Hindlll, the larger, about 3.1 kbp vector fragment isolated, and subsequently ligated with the 0.1 kbp long Eagl/ Hindlll fragment of pUR2653, which comprised the AFP coding sequence, thereby generating an construction intermediate with an unique Pstl site in the 3\'-coding part. The vector was digested with Pstl/Hindl l, the about 3.2 kbp fragment purified and ligated with the Pstl/Hindlll digested PCR product. After transformation into E.coli JM109 the plasmid was isolated and sequenced as described earlier. The complete about 0.25 kbp Eagl/ Hindlll fragment containing the two directly linked AFP coding parts, was then exchanged against small Eagl/Hindlll fragments in pUR2652 and pUR2653. Both plasmids, denoted pUR2676 and pUR2677, were sequenced as described to confirm the accurate assembly of the fragments (see Figure 7a and 8a).

To further expand the AFP copy number in the plasmid, a second PCR reaction can be executed, based on plasmid pUR2676 as template. Beside the ohgonucleotide AFPJ2JVA, which automatically creates a Stwl site behind the AFP coding sequence, a new 5\' PCR ohgonucleotide was synthesized, which allows the generation of a DNA fragment coding for two directly joined AFP coding parts, with an additional Hindi site overlapping with the first codon of the first AFP copy.

pUR2676 5\' GAT ACC GCC TGT GAT GCC GCC GCT GCC

3\' CTA TGG CGG ACA CTA CGG CGG CGA CGG II II II III III III III III III

AFPR3JV 5" AAACCGAATTC GTC GAC ACC GCC TGT GAT GCC GCC GCT GCG

_?CORI Hindi

After PCR this DNA fragment could be isolated and purified as described, digested with EcoRI and Hindlll and cloned into one of the commercially available usual E. coli cloning plasmids, eg. pTZ19R ex Pharmacia. From this plasmid the about 0.25 kbp Hincll/Stul fragment can be isolated and ligated behind the unique Stwl site present at the end of the second AFP coding sequence in pUR2676 and pUR2677 respectively, thereby producing the plasmids pUR2678 and pUR2679 (Figure 7b and 8b). This ligation can be repeated several times, thereby increasing the in each ligation round the copy number behind either the SUC2 sequence or the prepro-α-mating factor sequence with two.

EXAMPLE 7: Measurement α-galactosidase activity of pUR2652 S. cerevisiae transformants

From the Qiagen purified plasmid DNA of pUR2652, containing the α-galacto¬ sidase- AFP fusion under the control of the inducible GAL7 promoter and behind the invertase signal sequence about 5 μg was transformed to S. cerevisiae SU10 according to the method of Beggs et al. 1978. and plated onto YNB-plates containing histidine and uracil. Several of this transformants were incubated overnight in a 4 ml culture YNB, with 2 % glucose, ura and his. To test the induction of expression about 400μl of the overnight cultures were used to inoculate a 20 ml culture YP medium, containing 5 % galactose. After incubation at 30 °C for about 40 hours, the A660 was determined, the culture was centrifuged for 5 min with 4000 rpm and the α-galactosidase activity in the supernatant was measured. α-galactosidase activity was determined at 37°C using p-nitrophenyl α-D-galacto- pyranoside (PNPG, ex Sigma) as substrate in a 0.1 M sodium acetate buffer, pH 5.0. A solution of 22.22 mM PNPG, in acetate buffer was freshly prepared and 0.1 ml of an 1: 1000 in acetate buffer diluted supernatant solution was added to 0.9 ml pre-

warmed substrate solution. The mixture was incubated in a thermostatically controlled water bad for 5-10 minutes, and then stopped by addition of 2 ml 10% sodium carbonate solution. A blank was made as described below, by adding just 0.1 ml sodium acetate. The concentration of p-nitrophenolate was determined at 5 405 nm at room temperature. The difference between the molar absorptivity of p-nitrophenolate and P-nitrophenyl-α-galactopyranoside at 405 nm and pH12 was determined experimentally as 18.160 cm ² / μmol. Therefore the enzyme activity was calculated with the help of the following formula: {(At -Ab)/t} * 1.652 μmol per ml, where At is the absorbance at the incubation time t in minutes, and Ab the absor- 0 bance of the blank. One Unit was defined as the hydrolysis of a μmol of substrate in 1 min at 37°C and at a pH of 4.5. The specific activity of guar α-galactosidase is 100 U per mg protein.

For the quantitative assay of the α-galactosidase enzyme in the culture medium of S. cerevisiae SU10 cells containing pUR2652 different dilutions were used. The 5 expression level was calculated as mg α-galactosidase/L culture medium. As controls served a SU10 strain transformed with pUR2740, the parental plasmid without the AFP cassette and SU10 untransformed, to correct for the natural MEL1 expression level. A concentration of about 300 mg/L culture fluid was determined for different SU10 α-gal/AFP transformants, compared to 6 mg/1 for the untransformed SU10 0 and approximately 300 mg/1 for the SU10 with pUR2740, indicating the same amount of expression and secretion for the fusion protein as for α-galactosidase. Since both enzymatic measurements were based on the same amount of protein present, this also indicates an identical specific α-galactosidase activity for the fusion construct. 5

EXAMPLE 8. SDS-PAGE protein gelelectrophoresis and Western analysis of cell and culture fluid of SU10 cells containing either pUR2652 or pUR2653 48 hours incubated cultures were centrifuged for 5 min at 4000 rpm and cells and 0 supernatant were collected at stored at -20 °C. The cells were broken with glass beads and after isolation the cell-extract was centrifuged in an Eppendorf centrifuge for 5 min. The soluble cell fraction supernatant was stored at -20 °C. Denaturating

SDS polyacrylamide gel electrophoresis to separate the proteins was performed mainly as described by Laemmli, 1970. The Western blot analysis of the SDS-PAGE electrophoresis was performed principally as described by Towbin et al, 1979, by semi dry blotting. After blotting the Immobilon-P membrane was blocked with 5 % skimmed milk in 150 mM NaCl, 50 mM Tris/HCl pH 7.4 and afterwards incubated in 1 % skimmed milk in 150 mM NaCl, 50 mM Tris/HCl, 0.1 % Tween 20, pH 7.4 and antiserum against AFP-6 or α-galactosidase respectively. Both antisera were raised by immunization of rabbits with α-galactosidase purified from guar, and with AFP-6 obtained by Merriefield synthesis. Incubation was performed overnight at room temperature under soft agitation. Unabsorbed antibodies were removed by washing with the incubation buffer (3x 5 min) The membrane was incubated with the second antibody (Goat anti-rabbit IgG (H + L), Horseradish Peroxidase Conjugate; Biorad, Richmond) for 2 hours at room temperature. The enzyme was developed using 4-chloro-l-naphthol. For the transformant containing pUR2652 Western analysis with AFP-6 antisera revealed specific signals (see Figure 8b). Here, specific signals were detected within the soluble fraction of the yeast cell and in the culture fluid, whereby in both cases the apparent size of the obtained fusion-protein was about 46 kD, which is a little bit larger than α-galactosidase expressed in yeast (Overbeeke et al, 1987), and in good agreement with the result expected, due to the of nearly 4 kD structural information. Antiserum against α-galactosidase reacted specifically with the same protein bands, thereby verifying the expression of the α- galactosidase AFP fusion protein in yeast (data not shown). Both PAA Coomassie stain and immune specific stain revealed some minor degradation products. A somehow different situation was observed with the SU10 cells transformed with pUR2653. Here, neither in the Coomassie stain nor in the immune specific stain any signal could be detected within the medium (see Figure 9a and 9b), while the Western analysis of soluble cell fraction revealed two bands, one of them a cross- reacting host protein, which is also present in the untransformed yeast (Figure 9b, lane "c"), while the second, significantly smaller signal was only obtained in transformed yeast cells, presumably presenting the AFP. Whether the absence of AFP in the culture medium was the result of inhibition of secretion or rapid degradation remains to be elucidated.

EXAMPLE 9. Thermal hysteresis assay with culture fluid of cells containing of pUR2652 and pUR2653

In principle, this assay measures the difference between freeze- and melting point (de Vries, 1983) The range, in which this effect is measured is around 1 degree centigrade. In the described experiment substances were dissolved in water, soaked through capillary forces into micro-slides and quickly frozen. The samples were then placed into a controlled thermal ethanol bath at a temperature where the initial crystal nucleus is stable (visually controlled with Olympus stereo microscope at a 15 fold magnification). The temperature was then stepwise decreased with 0.1°C/min until the crystals started growing, this was defined as crystallization temperature. This process was continued for 2 minutes, and than the temperature was slowly increased in the same intervals. As soon as visually detectable melting occurred, the melting temperature was reached. As a positive control a sample chemically synthesized winter flounder peptide was used, -produced by Merriefield synthesis-, at a final concentration of 3% , while α-galactosidase, the carrier protein in the fusion situation served as negative control. The results are graphically summarized in Figure 10. The difference between both control substances was significant, hence, a difference of 0.1°C in freeze and melting point was considered as inactivated, compared to the 0.7 degree for the chemically synthesized AFP. The concentrated culture fluid of cells carrying the fusion gene expressed by pUR2652 revealed a hysteresis of 0,5 degree, which is similar to the 3% solution chemically synthesized AFP.

The effect obtained with the culture fluid of cells with plasmid pUR2653 was with a thermal hysteresis of 0.8°C at the same level than the pure 3% AFP solution.

EXAMPLE 10. Recrystallization assay of yeast cells containing pUR2652 lμl of solutions containing 30% sucrose and either 3% of pure chemically synthesized AFP or approx. 40% of freeze dried supernatant of culture fluid of yeast cells containing pUR2652 -final concentration- were placed onto microscope slides, covered with BDH 16 mm slides and sealed. The sample was set on a Linkam

THMS coldstage, connected with a Linkam CS 196 cooling system and controlled by a Linkam TMS 91 controller. The stage was subsequently crash cooled

(δ 99 °C/ min.) to -40 °C to produce a large amount of small sized ice crystals. The size of the ice crystals in the solution was recorded by a video camera. The stage was then rapidly raised to -5 °C and kept there for 1 hour. The growth of the ice crystals was followed visually and again registered by video recording. While the solution bearing no additives displayed the foreseen large crystals after incubation 9 (Figure 11a), the sample containing 3% AFP remains generally unchanged compared to the starting conditions, clearly indicating the growth inhibiting effect of AFP (see Figure lib). The solution containing about 40% of the freeze dried supernatant of pUR2652 displayed a different picture. Here, the crystals appeared to be significantly smaller and more needlelike (see Figure lie), indicating the even a fusion construct comprising not more than about 5% structural information of AFP is capable of restraining ice crystal growth.

LITERATURE mentioned in the specification

Achstetter, T., Nguyen-Juilleret, M., Findeli, A., Merkamm, M., Le ione, Y. 1992.

New signal peptide useful for the secretion of heterologous proteins from yeast and its application for synthesis of hirudin. Gene, 110, 25-31. Becker, D.M., Guarente, L., 1991. High efficiency transformation of yeast by electroporation. in Meth.in Enzymol.,194, 182-186

Beggs, J.D. 1978. Transformation of yeast by a replicating hybrid plasmid. Nature,

275, 104-109.

Betz, R., Duntze., W., Manney., T.R., 1981. Hormonal control of the gamentogenesis in the yeast Saccharomyces cerevisiae. Gamete research, 4, 571-584.

Bitter, G.A., Chen, K.K., Banks, A.R., Lai, P.H., 1984. Secretion of foreign proteins from Saccharomyces cerevisiae directed by α -factor gene fusions. Proc. Natl. Acad.

Sci. USA, 81, 5330-5334.

Caplan, S., Kurjan, J., 1991. Role of α-factor and the MFαl α-factor precursor in mating in yeast. Genetics, 127, 299-307.

Chakrabartty, A., Yang, D.S.C., Hew, C.L., 1989. Structure function relationship in the winter flounder antifreeze polypeptide: alteration of the component growth rates of ice by synthetic antifreeze polypeptides. J. Biol. Chem., 264, 11313-11316.

Chang, C.N., Matteucci, M., Perry, LJ., Wulf, JJ., Chen, C.Y., Hitzeman, RΛ., 1986. Saccharomyces cerevisiae secretes and correctly processes human interferon hybrid proteins containing yeast invertase signal peptides. Mol. Cell. Biol., 6, 1812-1819.

Chung, C.T., Niemela, S,L., Miller, R.H., 1989, One-step preparation of competent

E.coli: Transformation and storage of bacterial cells in the same solution. Proc. Natl.

Acad. Sci. USA, 86; 2172-2175. Davies, P.L., Hew., C.L.,1990. Biochemistry of antifreeze proteins. The FASEB journal ,4, 2460-2468.

DeVries, A.L., 1983, Antifreeze peptides and glycopeptides in cold water fishes.

Annu. Rev. Physiol., 45, 245-260.

Erhart, E., Hollenberg, CP. 1981. Curing of Saccharomyces cerevisiae 2μm DNA transformation. Curr. Genet., 3, 83-89.

Fourney, R.M., Yoshi, S.B., Kao, M.H., Hew, C.L., 1984. Heterogeneity of antifreeze polypeptides from the Newfoundland winter flounder Pseudopleuronectes americanus. Can. J. Zool. 62, 28-33.

Guisez, Y., Tison, B., Vanderkerkhove, J., Detmolder, J., Bauw, G., Hargeman, G., Fiers, W., Contreras, R, 1991. Production and purification of recombinant human interleukin-6 by the yeast Saccharomyces cerevisiae. Eur. J. Biochem., 198, 217-222.

Hitzemann, R.A., Chen, C.Y., Dowbwenko, D J., Renz. M.E., Lui, C, Pai, R.,

Simpson, NJ., Kohr, W.J., Singh, A., Chrisholm, V., Hamilton, R., Chan, C.N., 1990.

Use of heterologous and homologous signal sequences for the secretion of foreign proteins from yeast. Meth. Enzymol., 185, 421-440.

Hsiao, K. 1991. A fast and simple procedure for sequencing double stranded DNA with Sequenase. Nucl. Acids Res. 19, 2787.

Knight, C.A., DeVries, A.L., Oolman, L.D., 1984. Fish antifreeze proteins and the freezing and recrystallization of ice. Nature, 308, 295-296. Laemmli, U.K. 1970, Cleavage of structural proteins during the assembly of heads of bacteriophage T4. Nature, 227, 680-685.

Melnick L.M.,turner, B.G., Puma, P., Price-Tillotson, B., Salvateo, K.A., Dumais,

D.R., Moir, D.T., Broeze, RJ., Avgerniros G.C, 1990, Characterization of a non glycosylated single chain urinary plasminogen activator secreted from yeast. J. Biol.

Chem., 265, 801-807. Moir, D.T., Dumais, D.R., (1987). Glycosylation and secretion of human αl- antitrypsin by yeast. Gene, 56, 209-217.

Mueller, G.M., McKnown, R.L., Corotto, L.V., Hague, D., Warren, G J. 1991.

Inhibition of recrystallization in ice by chimeric proteins containing antifreeze domains. J.Biol. Chem.,266, no 12, 7339-7344. Nagai, K., Thφgersen, H.C, 1987. Synthesis and sequence specific proteolysis of hybrid proteins produced in Escherichia coli. Meth. in Enzymol., 153, 464-475.

Overbeeke, N., Fellinger., A ., Hughes, S.G., 1987. Production of guar α- galactosidase by hosts transformed with recombinant DNA methods. PCT

International WO 87/07641. Perlmann, D, Halvorson, H.O., Cannon, L.E., 1982. Presecretory and cytoplasmic invertase polypeptides encoded by distinct mRNAs derived from the same structural gene differ by a signal sequence. Proc. Natl. Acad. Sci. USA, 79, 781-785.

Peters, I.D., Hew, CL, Davies, P.I., 1989. Biosynthesis of winter flounder antifreeze pro-protein in E. coli. Protein Engineering, 3, 145-151. Rothstein, sj., Lahners, K.N., Lazarus, CM., Baulcombe, D.C, Gatenby, A.A. 1987.

Synthesis of wheat α-amylase in Saccharomyces cerevisiae. Gene. 55, 353-357.

Sambrook, J. Maniatis, T., Fritsch, E.F. 1989. Molecular cloning. A laboratory manual. Second edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, New

York. 1989. Sanger, F., Nicklen, S. and Coulson, A.R. 1977. DNA sequencing with chain- terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467.

Sleep, D., Belfleld, G.P., Ballamce, D.J., Steven, J., Jones,S., Evans, L.R., Moir, P.D.,

Goodey, A.R., 1991, Saccharomyces cerevisiae strains that overexpress heterologous proteins. Biotech., 9, 183-187. Steube, K., Chaudhuri, B., Marki, W., Merryweather, J.P., Heim, J., 1991. α-factor leader mediated secretion of recombinant human insulin like growth factor I from

Saccharomyces cerevisiae. Eur. J. Biochem. 198, 651-657.

Towbin, H., Staehelin, T., Gordon, J. 1979. Electrophoretic transfer of proteins from polyaciylamide gels to nitrocellulose sheets: Procedure and some applications. Proc.

Natl. Acad. Sci. USA 76, 4350-4354.

Verbakel, J.,M.,A., 1991. Heterologous gene expression in the yeast Saccharomyces cerevisiae. PhD thesis. Rijksuniversiteit Utrecht, The Netherlands.

Warren, GJ., Mueller, G.M., McKnown, R.L., Dunsmuir, P., 1990. Antifreeze polypeptides. PCT International WO 90/13571.

Yang, D.S.C, Sax, M., Chakrabartty, A., Hew, CL. 1988. Crystal structure of an antifreeze polypeptide and its mechanistic implications. Nature, 333, 232-237.

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