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EP0423264A1 - Proteine de recombinaison protegeant du choc du froid, sa production et son utilisation dans l'agriculture - Google Patents

Proteine de recombinaison protegeant du choc du froid, sa production et son utilisation dans l'agriculture

Info

Publication number
EP0423264A1
EP0423264A1 EP19900904543 EP90904543A EP0423264A1 EP 0423264 A1 EP0423264 A1 EP 0423264A1 EP 19900904543 EP19900904543 EP 19900904543 EP 90904543 A EP90904543 A EP 90904543A EP 0423264 A1 EP0423264 A1 EP 0423264A1
Authority
EP
European Patent Office
Prior art keywords
protein
gene
promoter
coh
temperature
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19900904543
Other languages
German (de)
English (en)
Inventor
Joel Goldstein
Stephen POLITT
Masayori Inouye
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rutgers State University of New Jersey
Rutgers Health
Original Assignee
University of Medicine and Dentistry of New Jersey
Rutgers State University of New Jersey
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Medicine and Dentistry of New Jersey, Rutgers State University of New Jersey filed Critical University of Medicine and Dentistry of New Jersey
Publication of EP0423264A1 publication Critical patent/EP0423264A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • 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
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

Definitions

  • FIG 1 (FIG 1) - Major Cold- Shock Protein Induction.
  • the autoradiogram in A wa ⁇ .subjected to scanning densitometry and the percent methionine-labeled cs7.4 protein in the whole cell was determined for each time interval.
  • Each time point on the graph is the quantitation for the percent methionine-labeled cs7.4 protein at the end of each thirty minute interval. For example, between 30 and 60 minutes (60 minute time point on the ordinate) , cs7.4 accounted for 10% of the total methionine-labeled protein in the cell at 10° C.
  • the 0 time point accounts for the five minute pulse at 37° C, which is described in A.
  • Figure 3 (FIG 3) Stability of cs7.4.
  • a cell culture growing at 37°C was transferred to 15°C. After 30 minutes, the culture was pulse-labeled with [ SS S] Translabel for 30 minutes . The culture was then chased with nonradioactive methionine and cysteine for various lengths of time indicated above the autoradiogram shown here. The samples were electrophoresed as described in FIG 1. The 37°C sample was prepared as described in FIG 2. The arrow indicates cs7.4.
  • a noteworthy characteristic of the cs7.4 protein of the invention is its stability at temperatures above the temperature range at which it was induced and synthesized. Such physiological temperature may range from about above 15°C to about 40°C, or higher.
  • the data in FIG 3 shows the protein to be stable after synthesis at 15°C for 20 hours, (only about 30% of the protein degraded) , and stable at 37° C (for at least 1.5 hours) .
  • the invention further provides a cold-induced cytoplasmic protein, designated cs7.4 which is stable at growth temperature of a microorganism, e. g. , E ⁇ coli.
  • the polypeptide has the following partial amino acid sequence SGKMTG(X)VKWFNADKGFGFI wherein X is leucine or isoleucine. Both isoleucine and leucine have been identified (64% and 36%, respectively) .
  • the invention includes either and both polypeptides .
  • he polypeptide of the invention is a 70 amino acid residue protein.
  • the calculated molecular weight is 7402 daltons and the calculated pi is 5.92.
  • the polypeptide is very hydrophylic, containing over 20% charged residues. Lysi ⁇ e residues make up 10% of the protein. No homology was detected with any other sequence in the NBRF data base.
  • the mechanism suggested requires that, after an antifreeze polypeptide induces local ordering of the ice lattice , the dipole moment from the helical structure dictates the preferential alignment of the peptide to the c-axis of the ice nuclei; shifts of the helical conformation can then take place and torsional movement of the side chains of the hydrophilic amino acids strenghtens the bonding of the protein with the ice surface.
  • polypeptide cs7.4 of the invention is the first antifreeze protein cold-induced in E ⁇ coli that can be produced by genetic engineering methods. Work on the secondary structure of cs7.4 would also open other possibilities . It can be postulated for instance , that the only portion of the polypeptide which has ⁇ -helix configuration would be essential for the antifreeze function; and likewise, that only the portion of the nucleotide sequence which encodes such polypeptide fraction would be essential for such antifreeze application. 15
  • the cloned 2.4 kb Hindlll fragment containing the gene for cs7.4 was isolated from pUC9 by digesting with Hindlll and separating on a 5% polyacrylamide gel. The fragment was then subcloned into M13. DNA sequencing was performed by the chain termination method (Sanger et al, 1977) . DNA sequencing was accomplished using [ 3S S] dATP and the enzyme, Sequenase, by the method provided by the manufacturer (United States Biochemical Corporation) .
  • the partial nucleotide sequence of the cloned Hindlll fragment includes the sequence encoding cs7.4 and the promoter therefor.
  • the nucleotide sequence encoding the polypeptide of the invention cspA includes the following sequence of 210 nucleotides.
  • cspA The corresponding amino acid sequence encoded by cspA is as follows .
  • the sequence is shown in FIG 6B , it contains an open reading frame beginning with an ATG codon at nucleotide 617 of the cloned Hindlll fragment and extending for 210 nucleotides ending with a TAA termination codon.
  • This open reading frame is the coding region of the gene herein designated cspA responsible for cs7.4 synthesis.
  • the invention includes within its scope the nucleotide sequence or any partial sequence thereof which codes for the polypeptide cs7.4 or a polypeptide having the properties of cs7.4 (functional equivalent) .
  • the invention also includes any equivalent nucleotide sequence wherein one or more codons have been substituted by certain other codons, which equivalent nucleotide sequence codes for the cs7.4 polypeptide, or a functional equivalent thereof.
  • the cold-shock protein would be expressed by the gene coding for it under the control of the promoter of the invention.
  • promoters other than the lac promoter, such as the trp, tac, promoter, lambda pL, ompF, opp, and other promoters may be used to regulate the expression of the gene coding for the desired protein.
  • promoter like GAL10 and others may be suitable.
  • the cspA promoter of the invention which is active at low temperatures, can be used to control the expression of a protein other than the cs7.4 cold-shock protein.
  • a protein other than the cs7.4 cold-shock protein can be used to control the expression of a protein other than the cs7.4 cold-shock protein.
  • This properly opens up yet other possibilities .
  • This may be of particular interest where a particular protein which would be useful but for the fact that it is enzymatically (e.g. , proteolytically) degraded at physiological temperatures , could be expressed at low temperatures at which it is less susceptible to enzyme degradation.
  • the cspA promoter of the invention has been used in a classic model to control the expression of ⁇ -galactosidase.
  • a plasmid (pKM005) (21) containing the lac Z structural gene without promoter was compared with the plasmid containing the cspA promoter on an 806 bp Hindlll-PvuII fragment (pJJG04) . See FIG 9.
  • the results show that the cspA promoter is capable of directing a heterologous gene to express a selected protein.
  • plasmid containing the cold-shock protein coding sequence and its regulatory elements it is siecessary to firstldentify and isolate the locus.
  • oligonucleotide probe a partial amino terminal sequence of the protein is obtained.
  • a 10 ml culture of IS-_ coli SB 221 (7) was grown to a density of approximately 2 X 10 s cells /ml at 37° C and transferred to 14° C for 4 hours. Cells were then harvested and fractionated for the soluble fraction as previously described (9) .
  • a trace of protein pulse-labeled for 30 minutes after shift to 15 °C as described above was then mixed with 250 ug of soluble fraction protein.
  • Two-dimensional electrophoresis was then performed with isoelectric focusing in the first dimension (ampholines pH 3-10, 1.5%; pH 6-8, 0.5%) and SDS- polyacrylamide gradient gel electrophoresis (10-18.4% acrylamid ⁇ , 2.7% crosslinking) in the second dimension according to the method of O'Farrell (15) .
  • Separated protein was electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (IVIillipore Corp .
  • PVDF polyvinylidene difluoride
  • niixed degenerate oligonucleotide probe for Southern blot analysis was made to match a short region of the amino acid sequence as shown below:
  • Hybridization was carried out according to Maniatis et al (26) with the following exceptions .
  • Both the prehybridization and hybridization solution contained by volume/ml solution: 0.1 ml 50 X Denhardt's, 0.2 ml 30 X NET, 0.5 ml 20% Dextran Sulfate, and 0.05 ml 10% SDS. These solutions are described in Inouye and Inouye, (19) .
  • the oligomer that was used for the probe is shown above.
  • the [ 32 P] -labeled probe was made according to Inouye and Inouye, (19) , and the prehybridization and the hybridization was carried out at 32° C.
  • the filter was washed and dried according to Inouye and Inouye, (19) .
  • the autoradiogram from Southern blot hybridization with the mixed oligonucleotide probe indicated at least one distinct band in each digest.
  • the Hindlll digest yielded one band with a size of 2.4 kb (See FIG 5) .
  • Hindlll fragment The 2.4 kb Hindlll fragment was isolated in the followin manner.
  • a Hindlll digest of chromosomal DNA was fractionated on a 0.7% agarose gel. Gel slices were then excised at every 0.5 cm from the top of the gel. Each gel slice was frozen at -20°C for at least 20 minutes and then centrif ⁇ ged in an Eppendorf tube for 10 minutes. This was repeated three times', the last time adding some lmM Tris, O.lmM EDTA (pH 7.5) before freezings and the supernatant was collected after each centrifugation. The samples were then phenol extracted three times, ether extracted, and ethanol precipitated.
  • the csp promoter was used to direct the synthesis of ⁇ - galaetosidase in E ⁇ coli from the plasmid pJJG04.
  • This plasmid was constructed as follows. The 2.4 kb Hindlll fragment containing the gene was digested with PvuII. The resultant 806 bp fragment was separated on 0.8% agarose gel, the band excised and the DNA recovered by electroelution using a salt-bridge electroelution apparatus manufactured by IB I, Inc. as per manufacturer's instructions.
  • yeast cells such as Saccharomyces cerevisiae with the structural gene of the invention or of all or part of the nucleotide sequence shown in FIG 6.
  • Basic techniques of yeast genetics, appropriate yeast cloning and expression vectors and transformation protocols are discussed in Current Protocols in Molecular Biology, Supplement 5 (1989) (23) which is specifically incorporated herein by reference.
  • vertebrate cell cultures may be transformed, with the structural gene of the invention or part thereof or with part or all of the nucleotide sequence shown in FIG 6.
  • an appropriate cell culture such as a COS-7 line of monkey fibroblasts.
  • Appropriate techniques for the transfection of DNA into eucaryotic cells are described in Current Protocols, Section 9 (also incorporated herein by reference) . Illustrated protocols are shown to work well with such cell ines as HeLa, BLAB/c 3T3, NIH 3T3 and rat embryo fibroblasts.
  • the invention contemplates nucleotide sequences which encode a protein which has biological properties of, or similar enough to be eseentially a functional equivalent, of the protein of the invention.
  • the invention contemplates a promoter sequence which performs essentially the same function as that described herein. The invention thus intends to cover and covers the functional equivalent of the functional elements described and taught herein.

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne une protéine de E. coli protégeant contre le choc du froid, un gène structurel codant pour celle-ci, un promoteur pour le gène et pour d'autres protéines. On a mis au point des séquences d'ADN comprenant le gène codant la protéine, le promoteur ainsi que d'autres éléments fonctionnels. L'invention concerne également des structures. On a envisagé des hôtes adaptés transformés ainsi que des plantes transgéniques. Elle concerne en outre diverses applications et divers procédés dans la synthèse de protéines.
EP19900904543 1989-02-13 1990-02-13 Proteine de recombinaison protegeant du choc du froid, sa production et son utilisation dans l'agriculture Withdrawn EP0423264A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US31033289A 1989-02-13 1989-02-13
US310332 1989-02-13

Publications (1)

Publication Number Publication Date
EP0423264A1 true EP0423264A1 (fr) 1991-04-24

Family

ID=23202035

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19900904543 Withdrawn EP0423264A1 (fr) 1989-02-13 1990-02-13 Proteine de recombinaison protegeant du choc du froid, sa production et son utilisation dans l'agriculture

Country Status (4)

Country Link
EP (1) EP0423264A1 (fr)
JP (1) JPH04500459A (fr)
CA (1) CA2009917C (fr)
WO (1) WO1990009447A1 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992019718A1 (fr) * 1991-05-03 1992-11-12 Smithkline Beecham Corporation PROMOTEURS REGULES A FAIBLE TEMPERATURE DANS $i(E. COLI)
US5981280A (en) * 1996-03-22 1999-11-09 The University Of Medicine And Denistry Of New Jersey Method and constructs for inhibiting protein expression in bacteria
ATE377647T1 (de) 1997-11-20 2007-11-15 Takara Bio Inc Durch kälte induzierbarer expressionsvektor
EP1452596A4 (fr) * 2001-11-19 2005-10-12 Riken Promoteurs reagissant aux contraintes de l'environnement et genes codant pour le facteur de transcription
SI1668141T1 (sl) * 2003-09-29 2013-04-30 Monsanto Technology, Llc Postopki za povečanje tolerance za sušo pri rastlinah in postopki za to
EP2281895B1 (fr) * 2003-09-29 2018-01-03 Monsanto Technology, LLC Procedes d'amelioration de tolerance au stress sur les plantes et compositions correspondantes
JP5279339B2 (ja) 2008-05-16 2013-09-04 タカラバイオ株式会社 逆転写反応用組成物
CN110467655B (zh) * 2019-08-14 2021-10-08 上海交通大学 一种蛋白质及其应用

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Publication number Priority date Publication date Assignee Title
US4464473A (en) * 1982-04-23 1984-08-07 The Regents Of The University Of California Ice nucleating microorganisms

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9009447A1 *

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Publication number Publication date
CA2009917A1 (fr) 1990-08-13
JPH04500459A (ja) 1992-01-30
WO1990009447A1 (fr) 1990-08-23
CA2009917C (fr) 2007-05-22

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