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WO1994003617A1 - Procede de production de peptides antigel - Google Patents

Procede de production de peptides antigel Download PDF

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Publication number
WO1994003617A1
WO1994003617A1 PCT/EP1993/001969 EP9301969W WO9403617A1 WO 1994003617 A1 WO1994003617 A1 WO 1994003617A1 EP 9301969 W EP9301969 W EP 9301969W WO 9403617 A1 WO9403617 A1 WO 9403617A1
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Prior art keywords
afp
gene
sequence
dna
encoding
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PCT/EP1993/001969
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English (en)
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Holger York Toschka
Johannes Maria A. Verbakel
Josina Wilhelmina Almkerk
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Unilever N.V.
Unilever Plc
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Priority to AU45719/93A priority Critical patent/AU4571993A/en
Publication of WO1994003617A1 publication Critical patent/WO1994003617A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/047—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with yeasts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)

Definitions

  • the invention relates to transforming host cells, in particular yeast cells, for expression and secretion of peptides which inhibit ice crystal growth.
  • Antifreeze proteins 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.
  • the HPLC-6 and the HPLC-8 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,).
  • the 37 amino acids long molecule contains three 11 amino acids tandem repeats, with mainly alanine amino acids that favour a single ⁇ 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).
  • ⁇ -factor is a 13-amino acid peptide that is encoded by two unlinked structural genes, MF ⁇ l and MF ⁇ 2. 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 ⁇ -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 ⁇ mating factor is released into the surrounding medium.
  • ⁇ leader segment mediated secretion from 5. cerevisiae is described in the literature for several different proteins and peptides, for example a consensus ⁇ -IFN, ⁇ -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 ⁇ -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).
  • 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).
  • a desired gene encoding a protein active in the inhibition of ice crystal growth is placed in frame as a fusion construct behind the ⁇ -galactosidase gene of Cyamopsis tetragonoloba.
  • the new chimeric gene is placed under the control of the strong inducible GAL7 promoter.
  • the whole gene is placed behind the invertase signal sequence.
  • a gene encoding the desired anti ⁇ freeze peptide is directly expressed under the control of the inducible GAL7 promoter.
  • the gene coding for the mature AFP is separated by the pre-pro sequence of the a mating factor from the promoter.
  • a-gal synthetic gene coding for ⁇ -galactosidase from Cyamopsis tetragonoloba.
  • amp r. ⁇ -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 ⁇ - galactosidase gene represents the SUC2 signal sequence
  • afp DNA coding for one copy of a synthetic AFP gene
  • the thicker line between ⁇ -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.
  • GAL7-ISS Gal7 promoter and SUC2 signal sequence
  • FIG. 3 a) Schematic presentation of plasmids pUR2660 containing 2 copies of AFP separated by KREA. b) plasmid pUR2665, containing 3 copies of
  • 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.
  • Figure 5 Schematic presentation of plasmid pUR2674 containing the fusion between ⁇ -galactosidase and 5 copies of AFP, separated by the last 7 amino acids of the ⁇ -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 ⁇ -mating factor sequence.
  • Figure 8. a) SDS gel analysis. b) Western analysis of soluble cell fractions and culture fluids of yeast cells containing pUR2652.
  • ⁇ -gal purified ⁇ -galactosidase
  • c soluble cell fraction
  • s supernatant of cell cultures
  • 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.
  • ⁇ -gal purified ⁇ -galactosidase
  • 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
  • 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 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
  • 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,
  • the "host organism” can be a plant or animal cell or a microorganism.
  • 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).
  • the DNA construct contains a fused gene comprising at least part of the ⁇ -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 ⁇ - 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.
  • 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.
  • a constitutive promoter is preferable under conditions where it is advantageous to express the desired gene in all growth stages.
  • the gene should comprise or be preceded by an "ATG start codon" for starting proper translation of the mRNA.
  • DNA signal sequence can be used for example the invertase signal sequence of S. cerevisiae or the DNA pre-sequence of the ⁇ -mating factor of 5. cerevisiae.
  • DNA construct can also comprise the pro-sequence of the ⁇ -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.
  • a signal sequence need not be present. Of course the AFP need then not be collected from the host organism.
  • a signal sequence is preferred for obtaining efficient secretion.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 (mat ⁇ , 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
  • Transformants were recovered on selective YNB-plates (0.67% YNB, 2% glucose,
  • GAL7 promoter Restriction enzymes and DNA modification enzymes were applied as recommended by the supplier.
  • Plasmid pUR2652 was constructed to obtain a C-terminal fusion of AFP to ⁇ -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.
  • 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).
  • 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.
  • IEGR amino acid sequence for the blood factor Xa
  • This construction allowed the later release the pure AFP from the fusion protein.
  • the nucleotides for the C-terminus of ⁇ -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.
  • the S ⁇ cl/H dlll fragment of pUR2740 comprising the 3' end of the GAL7 promoter, the SUC2 signal sequence and the complete synthetic ⁇ -galactosidase gene (Verbakel, 1991) was subcloned into pTZ19R
  • oligonucleotides 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 ⁇ l 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 20 ⁇ l, 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.
  • 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 lO ⁇ l of all five duplex mixtures were pooled and incubated in a final volume of lOO ⁇ l, containing 50 mM Tris/ ⁇ Cl, p ⁇ 7.5, 8 mM MgC12, 8 mM DTT, and 40 ⁇ g/ml gelatine and 10 U of DNA ligase, for two hours at 20°C. The ligation mix was then precipitated, and redissolved in 30 ⁇ l of TE-buffer.
  • AFPOl 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 Hindl ⁇ l 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).
  • This synthetic genes simply generates an in frame transition between the coding part of AFP and the E ⁇ gl site at the end of the prepro-sequence in vector PSY16, a 2 ⁇ m derived shuttle vector for leucine complementation, where the C-terminal end of the natural prepro ⁇ 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.
  • 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.
  • the vector pUR2553 was linearized with E ⁇ gl, 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).
  • 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 ⁇ helical structure.
  • 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 E ⁇ gl/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.
  • AFP-6 Another possibility for increased AFP peptide expression in yeast is the repeated fusion of AFP-6 behind the carrier protein ⁇ -galactosidase, separated by internal spacers originating from the intervening sequences of the ⁇ -mating factor precursor molecule.
  • 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 ⁇ -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 ⁇ -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.
  • 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.
  • PCR amplification of the template was carried out in a Perkin Elmer Cetus DNA Thermal Cycler.
  • the reaction was carried out in 100 ⁇ l 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 ⁇ g 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.
  • 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]dATP ⁇ S (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.
  • Arg 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.
  • PCR primer AFPPRIJVA 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 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).
  • 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.
  • 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).
  • a second PCR reaction can be executed, based on plasmid pUR2676 as template.
  • 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.
  • AFPR3JV 5" AAACCGAATTC GTC GAC ACC GCC TGT GAT GCC GCC GCT GCG
  • 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- ⁇ -mating factor sequence with two.
  • EXAMPLE 7 Measurement ⁇ -galactosidase activity of pUR2652 S. cerevisiae transformants
  • ⁇ -galactosidase activity was determined at 37°C using p-nitrophenyl ⁇ -D-galacto- pyranoside (PNPG, ex Sigma) as substrate in a 0.1 M sodium acetate buffer, pH 5.0.
  • PNPG p-nitrophenyl ⁇ -D-galacto- pyranoside
  • 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.
  • a concentration of about 300 mg/L culture fluid was determined for different SU10 ⁇ -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 ⁇ -galactosidase. Since both enzymatic measurements were based on the same amount of protein present, this also indicates an identical specific ⁇ -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 ⁇ -galactosidase respectively.
  • Both antisera were raised by immunization of rabbits with ⁇ -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).
  • 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.
  • 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.
  • 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 l ⁇ l 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
  • the solution containing about 40% of the freeze dried supernatant of pUR2652 displayed a different picture.
  • 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.
  • Saccharomyces cerevisiae secretes and correctly processes human interferon hybrid proteins containing yeast invertase signal peptides. Mol. Cell. Biol., 6, 1812-1819.
  • E.coli Transformation and storage of bacterial cells in the same solution. Proc. Natl.

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Abstract

Procédé de production de peptides antigel (AFP), consistant à exprimer chez un organisme hôte, de préférence une levure, un gène codant des AFP régulés par un promoteur puissant, par exemple un promoteur inductible GAL7 ou constitutif GAPDH de Sacchromyces cerevisiae. Le gène d'AFP peut être précédé par une séquence de signaux destinés à la sécretion de la protéine, par exemple, la séquence de signaux d'invertase ou la préséquence du facteur d'α-apariment de S. cerevisiae. La partie non-AFP du gène fusioné peut être une partie du gène α-galactosidase de Cyamopsis tetragonoloba. La structure du gène peut comprendre un site de traitement que l'on peut utiliser pour retrancher au moins un AFP du reste de la protéine. Le gène d'AFP peut être présent sous la forme d'une répétition en tandem de gènes d'AFP. L'invention concerne également des AFP produits tels que ci-dessus décrit, et l'utilisation de ces AFP pour inhiber la croissance de cristaux de glace, par exemple, dans de la crème glacée et d'autres produits alimentaires, ou d'autres matières biologiques pouvant être congelées. En outre, l'invention concerne une levure présentant une meilleure tolérence à la congélation, contenant et/ou envelopée par des AFP produits comme ci-dessus décrit, laquelle peut être utile dans la production de pâtes congelées ou d'autres compositions pouvant être congelées.
PCT/EP1993/001969 1992-07-29 1993-07-22 Procede de production de peptides antigel WO1994003617A1 (fr)

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Cited By (21)

* Cited by examiner, † Cited by third party
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WO1996011586A1 (fr) * 1994-10-12 1996-04-25 Hsc Research And Development Limited Partnership Preparation d'aliments fermentes glaces en utilisant des micro-organismes exprimant un polypeptide antigel
WO1997002343A1 (fr) * 1995-07-05 1997-01-23 Unilever Plc Peptide de recombinaison
FR2751513A1 (fr) * 1996-07-26 1998-01-30 Unilever Nv Produit alimentaire congele
EP0843010A1 (fr) 1996-11-19 1998-05-20 Unilever Plc Polypeptides antigel de carotte
WO1998041107A1 (fr) * 1997-03-14 1998-09-24 Unilever N.V. Produit alimentaire congele
US6017574A (en) * 1995-06-07 2000-01-25 The Pillsbury Company Method of making frozen compositions
US6090917A (en) * 1996-07-26 2000-07-18 Good Humor-Breyers Ice Creams Frozen food product
US6200622B1 (en) 1997-03-14 2001-03-13 Good Humor - Breyers Ice Cream, Division Of Conopco Frozen food product
US6793952B2 (en) 1996-07-26 2004-09-21 Good Humor-Breyers Ice Cream, Division Of Conopco, Inc. Frozen food product
US6852841B1 (en) 1998-01-22 2005-02-08 Good Humor-Breyers Ice Cream, Division Of Conopco, Inc. Frozen food product
US7175865B2 (en) 2004-05-12 2007-02-13 General Mills Marketing, Inc. Method of producing frozen dough, and related products
EP1917865A1 (fr) 2006-10-20 2008-05-07 Nestec S.A. Peptides structurant la glace d'origine laitière
EP2048236A1 (fr) * 2003-12-23 2009-04-15 Novozymes Biopharma DK A/S Technique d'expression genique
WO2012026339A1 (fr) 2010-08-25 2012-03-01 株式会社カネカ Inhibiteur de cristallisation de glace dérivé de basidiomycètes
US8178151B2 (en) 2005-12-21 2012-05-15 Conopco, Inc. Frozen aerated confection
US8252551B2 (en) 2003-12-23 2012-08-28 Novozymes Biopharma Dk A/S 2-micron family plasmid and use thereof
WO2012121172A1 (fr) 2011-03-04 2012-09-13 株式会社カネカ Inhibiteur de la cristallisation de glace issu de graines végétales
US8969064B2 (en) 2003-12-23 2015-03-03 Novozymes Biopharma Dk A/S Gene expression technique
US8993030B2 (en) 2005-09-23 2015-03-31 Conopco Low pH aerated products
US9005690B2 (en) 2005-09-23 2015-04-14 Conopco, Inc. Aerated products with reduced creaming
US9115349B2 (en) 2008-10-16 2015-08-25 Conopco, Inc. Hydrophobin solution containing antifoam

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VERBAKEL J.M.A.: "Heterologous gene expression in the yeast Saccharomyces cerevisiae", PHD THESIS . RIJKSUNIVERSITEIT, 1 May 1991 (1991-05-01), UTRECHT, THE NETHERLANDS, pages 51 - 120 *

Cited By (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996011586A1 (fr) * 1994-10-12 1996-04-25 Hsc Research And Development Limited Partnership Preparation d'aliments fermentes glaces en utilisant des micro-organismes exprimant un polypeptide antigel
AU711640B2 (en) * 1994-10-12 1999-10-21 Hsc Research And Development Limited Partnership Preparation of frozen fermented foods using antifreeze polypeptide-expressing microorganisms
US6017574A (en) * 1995-06-07 2000-01-25 The Pillsbury Company Method of making frozen compositions
WO1997002343A1 (fr) * 1995-07-05 1997-01-23 Unilever Plc Peptide de recombinaison
EP1614753A3 (fr) * 1995-07-05 2006-07-19 Unilever Plc Expression de peptides antigel de poissons marins dans un organisme de qualité alimentaire et son utilisation dans des produits alimentaires
EP1614753A2 (fr) 1995-07-05 2006-01-11 Unilever Plc Expression de peptides antigel de poissons marins dans un organisme de qualité alimentaire et son utilisation dans des produits alimentaires
US6914043B1 (en) 1995-07-05 2005-07-05 Good Humor - Breyers Ice Cream, A Division Of Conopco, Inc. Frozen food products comprising anti-freeze protein (AFP) type III HPLC 12
AU723039B2 (en) * 1995-07-05 2000-08-17 Unilever Plc Expression of ocean fish antifreeze peptide in a food grade organism and its application in food products
US6793952B2 (en) 1996-07-26 2004-09-21 Good Humor-Breyers Ice Cream, Division Of Conopco, Inc. Frozen food product
FR2751513A1 (fr) * 1996-07-26 1998-01-30 Unilever Nv Produit alimentaire congele
US6090917A (en) * 1996-07-26 2000-07-18 Good Humor-Breyers Ice Creams Frozen food product
AU726699B2 (en) * 1996-07-26 2000-11-16 Unilever Plc Frozen food product containing heat stable antifreeze protein
US6156880A (en) * 1996-07-26 2000-12-05 Good Humor-Breyer's Ice Cream, Division Of Conopco, Inc. Frozen food product
US6162789A (en) * 1996-07-26 2000-12-19 Good Humor-Breyers Ice Cream, Division Of Conopco, Inc. Frozen food product
WO1998004148A3 (fr) * 1996-07-26 1998-04-23 Unilever Nv Produit de confiserie surgele
GB2315753B (en) * 1996-07-26 2001-06-13 Unilever Plc Frozen confectionery containing anti-freeze proteins
WO1998022591A3 (fr) * 1996-11-19 1998-07-30 Unilever Nv Polypeptides antigel tires de la carotte
US6797690B1 (en) 1996-11-19 2004-09-28 Good Humor — Breyers Ice Cream, division of Conopco, Inc. Carrot antifreeze polypeptides
EP0843010A1 (fr) 1996-11-19 1998-05-20 Unilever Plc Polypeptides antigel de carotte
US6200622B1 (en) 1997-03-14 2001-03-13 Good Humor - Breyers Ice Cream, Division Of Conopco Frozen food product
WO1998041107A1 (fr) * 1997-03-14 1998-09-24 Unilever N.V. Produit alimentaire congele
US6852841B1 (en) 1998-01-22 2005-02-08 Good Humor-Breyers Ice Cream, Division Of Conopco, Inc. Frozen food product
EP2048236A1 (fr) * 2003-12-23 2009-04-15 Novozymes Biopharma DK A/S Technique d'expression genique
US8252551B2 (en) 2003-12-23 2012-08-28 Novozymes Biopharma Dk A/S 2-micron family plasmid and use thereof
US8969064B2 (en) 2003-12-23 2015-03-03 Novozymes Biopharma Dk A/S Gene expression technique
US9057061B2 (en) 2003-12-23 2015-06-16 Novozymes Biopharma Dk A/S Gene expression technique
US7175865B2 (en) 2004-05-12 2007-02-13 General Mills Marketing, Inc. Method of producing frozen dough, and related products
US8993030B2 (en) 2005-09-23 2015-03-31 Conopco Low pH aerated products
US9005690B2 (en) 2005-09-23 2015-04-14 Conopco, Inc. Aerated products with reduced creaming
US8178151B2 (en) 2005-12-21 2012-05-15 Conopco, Inc. Frozen aerated confection
EP1917865A1 (fr) 2006-10-20 2008-05-07 Nestec S.A. Peptides structurant la glace d'origine laitière
US9115349B2 (en) 2008-10-16 2015-08-25 Conopco, Inc. Hydrophobin solution containing antifoam
WO2012026339A1 (fr) 2010-08-25 2012-03-01 株式会社カネカ Inhibiteur de cristallisation de glace dérivé de basidiomycètes
US8734672B2 (en) 2010-08-25 2014-05-27 Kaneka Corporation Ice crystallization inhibitor derived from basidiomycete
WO2012121172A1 (fr) 2011-03-04 2012-09-13 株式会社カネカ Inhibiteur de la cristallisation de glace issu de graines végétales

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