WO1990009447A1 - 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 Download PDF

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WO1990009447A1
WO1990009447A1 PCT/US1990/000776 US9000776W WO9009447A1 WO 1990009447 A1 WO1990009447 A1 WO 1990009447A1 US 9000776 W US9000776 W US 9000776W WO 9009447 A1 WO9009447 A1 WO 9009447A1
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protein
gene
promoter
coh
temperature
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PCT/US1990/000776
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English (en)
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Joel Goldstein
Stephen N. Pollitt
Masayori Inouye
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University Of Medicine And Dentistry Of New Jersey
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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
    • 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

  • This invention relates generally to the field of biotechnology, more specifically to a novel and valuable protein which on the basis of N( evidence to date, is believed to be capable of protecting cells, e. g. , plant cells against the adverse and injurious effects of low temperature (an antifreeze protein) and is stable at physiological temperatures .
  • the invention also relates to methods of making same.
  • the invention further relates to a novel promoter which controls the expression of the gene encoding the protein or of heterologous proteins.
  • the invention further relates to a cold shock structural gene the expression of which is capable of being regulated by a heterologous promoter.
  • the invention relates further to transformed competent hosts, contemplates transgenic plants which can express the antifreeze protein and to several other ⁇ useful applicatio ⁇ s in agriculture and other fields.
  • Damage to crops by frost is one of the leading causes of loss in agricultural output due to the natural phenomenon of weather variability in the world. It has been estimated that from 5 to 15% of the gross world agricultural product may be lost to frost damage in one year. In some regional areas the loss may approach 100%. The yearly economic crop loss due to frost damage in the United States has been reported tp be greater than $5 billion. Most of this damage is induced by freezing initiated by certain species of natural ice -nucleating bacteria, such as species of Pseudomonas , such as syringae, coronaf aciens , pisi, tabaci, or fluorescens , Xanthomonas such as translucens , or Erwinia such as herbicola.
  • Pseudomonas such as syringae, coronaf aciens , pisi, tabaci, or fluorescens
  • Xanthomonas such as translucens
  • Erwinia
  • Such natural epiphytic bacteria on the plant surface can promote the nucleation and formation of ice crystals at temperatures slightly below 0°C.
  • Such ice nucleation capable (INA "*” ) bacteria are responsible for the frost damage on a wide variety of important agricultural crops, such as corn, soybeans, wheat, tomatoes, deciduous fruit trees such as pears, almond, apple, cherry, and many subtropical plants such as citrus and avocado .
  • the treatment of plants with bacterial strains is subject to regulatory and public response in the United States and elsewhere, and the present invention suggests in one of its important embodiments an alternative to applying non-ice nucleation bacterial strains .
  • the invention provides a protein which is thought to function on the basis of evidence available, as an antifreeze protein; also the invention contemplates the transformation of plants to render them less susceptible or resistant to low temperature by incorporating the DNA sequence carrying the gene into recipient crop host cells which will then synthesize the antifreeze protein of the invention, or one of similar antifreeze properties.
  • the ability to control a gene encoding a protein of commercial value such that it is produced only after the temperature has been reduced below the normal growth temperature would allow the expression, of the target protein to proceed at optimal temperature with no ill effects on the desired physiological activity of the protein.
  • United States Patent No. 4,766, 077 (1988) deals with iee-nucleation deficient (INA-) microorganisms which have induced non-reverting mutations within a genomic DNA sequence (INA gene) which encodes for polypeptide(s) responsible for ice-nucleation activity (INA) .
  • the INA- microorganisms disclosed are applied to a plant part so that they become established prior to the colonization of the plant by the INA " " microorganisms .
  • Treatment in accordance with the patent is apparently the subject of a notice of an application for permission with the United States government to release Pseudomonas syringae pv. syringae and Erwinia herbicola carrying in vitro generated deletions of all or part of the genes involved in icemucleation.
  • U. S. Patent No. 4,375 ,734 (1983) also deals with an ice nucleation- inhibiting composition using non-phytotoxic virulent bacteriophages which are species specific to the ice-nucleating bacteria normally present on plants.
  • a particularly useful strain is a virulent bacteriophage strain, Erh 1, which is species specific to Erwinia herbicola.
  • U. S. Patent No. 4,464,473 (1984) provides for the isolation of DNA segments encoding for ice nucleation activity, which DNA segments may then be introduced into an appropriate vector.
  • the ice-nucleating microorganisms can be used for preventing super cooling of water in various commercial applications.
  • U. S. Patent No. 4,161,084 (1979) provides for a method for reducing the temperature at which freezing takes place in plants to reduce frost damage. This is performed by the addition of non-ice-nucleating bacteria to plants prior to the onset of freezing temperature and preferably while the plants are in their seedling stage.
  • protein synthesis was reported to decrease for one hour and then cease.
  • the accumulation of 70s ribosomes that were found after such a temperature shift has been interpreted to indicate a block in initiation of translation.
  • a shift to 10° C results in a growth lag of 4 hours followed by renewed growth.
  • Such proteins are low molecular weigh ⁇ proteins commonly found in high concentrations in the serum, of pola dwelling marine fishes and in the hemolymph of insects which winter in subfreezing climates .
  • FIG 1 shows the major cold-shock protein induction.
  • the arrow on the right indicates the induced major cold-shock protein, cs7.4.
  • FIG 2 shows transient induction of cs7.4.
  • the numbers indicated are as follows: 1, pulse-labeled from 0 to 30 minutes after temperature shift; 2, 30 to 60 minutes; 3, 60 to 90 minutes; 4, 90 to 120 minutes; 5, 120 to 150 minutes; 6, 150 to 180 minutes.
  • the arrows are described in FIG 1.
  • FIG 3 shows the stability of cs7.4.
  • the arrow indicates cs7.4.
  • FIG 4 shows the 2-dimensional gel utilized in purification of cs7.4.
  • FIG 5 shows a Southern Blot analysis for cs A.
  • FIG 6 A shows the DNA sequence of cspA (restriction map and sequencing strategy) .
  • FIG 6B shows the partial nucleotide sequence of the cloned Hindlll fragment.
  • the region encoding cspA and the corresponding amino acid sequence of cs7.4 are shown in bold type .
  • the underlined AGG is probably the Shine-Delgarno sequence. Regions underlined with half an arrow indicate inverted repeats.
  • FIG 7 is a schematic representation of a plasmid pJJGOl carrying the cspA gene (shown by arrow) .
  • FIG 8 is a schematic representation of pJJG12.
  • FIG 9 is a schematic representation of pJJG04.
  • Methods, systems and compositions are provided for genetically transforming microorganisms, particularly bacteria, to produce genotypical capability, particularly to produce "cold-shock” or “antifreeze” and other proteins.
  • segments of DNA that contain signals that direct the proper binding of the RNA polymerase holoenzyme and its subsequent activation to a form capable of initiating specific RNA transcription.
  • the promoter which is described herein is cold-induced and capable of controlling the expression of the cspA gene which encodes a cold- shock or antifreeze or other proteins . Practical applications are referred to hereinafter.-rison
  • Cold- shock or antifreeze proteins particularly a cold-shock protein of E ⁇ _ coli designated cs7.4.
  • the invention provides a novel polypeptide which is synthesized in E ⁇ coli in response to a decrease of the temperature below ambient, or physiological growth temperature.
  • the polypeptide (or protein) is a 7.4 kdal protein induced under cold-shock and has been designated as "cs7.4" .
  • polypeptide is stable at temperatures above the cold temperature at which it was induced.
  • the invention also provides the gene encoding the cs7.4 protein.
  • the invention provides further the cold-induced promoter which controls the expression of the gene encoding cs7.4 and which promoter also is capable of initiating transcription of proteins other than cs7.4 in response to a shift in temperature below the microorganism growth temperatures .
  • the invention also provides a system for expressing the gene encoding the antifreeze protein under the direction of a promoter other than the. promoter of the invention.
  • the invention further provides various DNA constructs , including cloning vectors, e. g. , plasmids which contain the promoter, the structural gene and other necessary functional DNA elements , and transformed hosts .
  • cloning vectors e. g. , plasmids which contain the promoter, the structural gene and other necessary functional DNA elements , and transformed hosts .
  • the invention also contemplates various applications of the products of the invention in the field of preventing or alleviating the injurious or lethal effects of low temperature, e.g. , freezing of microbiological and plant cells.
  • the protein of the invention be used as an "antifreeze" compound, for instance on crops; or that an innocuous microorganism transformed with the gene or portions thereof encoding for cs7. protein of the invention be used in agricultural or other applications .
  • DNA containing a sequence encoding the cs7.4 protein be transferred to crop host cells to produce there the cs7.4 antifreeze protein, and thus protect the crop from frost injury.
  • the invention provides a cold-shock induced promoter (the "native" promoter) which is capable of regulating the expression of a cold-shock induced gene encoding an antifreeze protein.
  • the invention provides further a promoter ("native") which is capable of controlling the expression of a gene encoding an antifreeze protein or another protein at temperature below physiological temperatures .
  • a promoter which is capable of controlling the expression of a gene encoding an antifreeze protein or another protein at temperature below physiological temperatures .
  • the invention also provides for the expression of a gene encoding an antifreeze protein under the control of a heterologous (non-native) promoter at physiological temperatures .
  • the invention further contemplates that having further elucidated the promoter sequence, the DNA sequence of the promoter can be generated synthetically; such promoter will be useful to regulate the expression of proteins at low temperatures, i.e. , temperatures below physiological temperature .
  • the promoter can be "uncoupled” in the sense that it can be used without the native structural gene and conversely, the structural gene can be controlled by a heterologous promoter.
  • FIG 1 (FIG 1) - Major Cold- Shock Protein Induction.
  • FIG 2 A and 2B (FIG 2 A and FIG 2B) - Transient Induction -of cs7.4.
  • 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.
  • FIG 4 (FIG 4) - 2-dimensional Gel Utilized in Purification of cs7.4.
  • a cell culture growing at 37° C was transferred to 14° C for 4 hours.
  • the culture was then harvested, fractionated, and the cytoplasmic f raction was subjected to 2-dimensional gel electrophoresis .
  • the first dimension is isoelectric focusing and the second dimension is SDS-polyacrylamide gel electrophoresis.
  • the gel was electroblotted onto a PVDF membrane which was stained with Coomassie Blue dye. The arrow indicates cs7.4.
  • FIG 5 (FIG 5) - Southern Blot Analysis for cspA.
  • E. coli chromosomal DNA was digested with various restriction enzymes and the DNA was transferred from an agarose gel to nitrocellulose paper. Hybridization was carried out using the degenerate probe described in the text, and the autoradiogram is shown here.
  • the restriction digests are as follows: S, Sail; P, PstI; B , BamHI; E, EcoRI; H, Hindlll. indicates lambda DNA digested with Hindlll, which was used as a size standard. Chromosomal DNA digested with Hindlll was also fractionated by agarose gel electrophoresis, and fractions 1 through 12 are shown.
  • FIG 6 A and 6B (FIG 6 A and 6B) - DNA Sequence of cspA.
  • the invention provides a method for producing a "c ⁇ ld-shock" protein, a promoter therefor and various constructs.
  • the gene encoding the cold-shock protein is expressed under the regulation of a heterologous promoter.
  • the cold-shock induced promoter is used to control the synthesis of a heterologous protein in response to lowering of the growth temperature .
  • the method of the invention to induce and produce the cs7.4 protein of the invention comprises growing in a nutrient rich medium at an exponential rate an appropriate microorganism, for instance an E-_ coli, to a desired growth density at physiological growth temperature for the particular microorganism.
  • an appropriate microorganism for instance an E-_ coli
  • such temperature may be in the range of about 10° to about 50° C, preferably in the range of 20° to about 40° C.
  • Each microorganism is known to have its optimum growth temperature; for E. coli raising the temperature above about 40° C or lowering it below 20° C results in progressively slower growth, until growth ceases, at the maximum temperature of growth, about 49° C , or the minimum, about 8°C .
  • the tempe ure is rapidly shifted to a lower temperature above about 10° and below about 20° C, preferably below about 20° C and above about 8°C. Lower temperatures generally do not sustain practical growth rates . If desirable , a shift to lower temperature but above the temperature at which no growth of the microorganism takes place may also be performed.
  • the culture is grown in the lower temperature range for the appropriate period of time for optimum production of the polypeptide of the invention.
  • the kinetics of polypeptide induction are followed by appropriate method such as pulse labeling with radioactive methionine, harvesting the culture, processing and separation by two-dimensional gel electrophoresis and determining by autoradiography the amount of protein synthesized .
  • the protein of the invention is not synthesized at physiological growth temperature at which the microorganism normally grows, in this case the E-_ coli; the polypeptide is synthesized in the lower temperature range. Sudden induction of the synthesis of the polypeptide takes place within approximately the first 30 minutes after temperature shift to 10°C or 15°C. Maximal induction and rate of synthesis is temperature dependent after temperature shift. After shift to 15° C, maximal synthesis is attained at 30-60 minutes post temperature shift; maximal rate of synthesis is approximately 13.1% of total protein synthesis. See FIGS 2A and 2B . Shift to 10° C gives a maximal rate of the synthesis of approximately 8.5% of total protein synthesis at 60 to 90 minutes post-shift.
  • Adjustment of the temperature therefore allows for adjusting the rate of synthesis and/ or yield of the polypeptide as being suited for the objective of the invention.
  • the rate of synthesis thereof drops off, ultimately reaching a fraction, e. g. , about a fifth of the maximum in the case of the culture shifted to 15° C and about three-fifths of the maximum in the case of the culture shifted to 10° C.
  • 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 stability of the protein of the invention at physiological temperatures has important practical applications. It permits synthesis of the protein at physiological temperatures with a promoter other than the promoter of the invention. It also facilitates applications of the protein in agricultural formulations on crops at ambient temperatures before the protein starts functioning in its freezing or frost damage prevention role. Other elements of the invention will be described hereinafter.
  • the promoter of the invention is believed to be located on the cloned Hindlll fragment between nucleotides 1 and 605.
  • the first 997 bp of the cloned Hindlll fragment contains all the necessary elements of the functional gene for regulated expression including the ribosome binding sites .
  • the promoter of the invention is activated at reduced temperature and directs transcription of the gene of the invention.
  • the promoter of the invention is cold-inducible in vivo and is recognized in vivo by RNA polymerase .
  • a cold activated inducer induces expression from the promoter at lower temperatures.
  • a cold sensitive repressor may be inactivated at cold temperatures resulting in expression of the gene.
  • the gene may be controlled by a cold induced alternate (other than a standard Bke the ⁇ "70 factor) sigma factor which would allow the RNA polymerase to recognize a novel promoter sequence upstream of the structural gene.
  • Other mechanisms may be postulated, especially if one considers that the transformed host need not be a member of the Enterobacteriaceae" family.
  • 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.
  • ATGTCCGGTi AAATGACTGGTATCGTAAAATGGTT ( _ ⁇ -ACGCTGACAAAGGCTTCGGCTTC ⁇ TCaCTCCTGAC GATGGCTCTAAAGATGTGTTCGTA ( - ⁇ CTTCTCTGCTATCCAGAACGATGGTTACAAATCTCTGGACGAAGGT CAGAAAGTGTCCTTCACCATCGAAAGCGGCGCTAAAGGCCCGGCAGCTGGTAACGTAACCAGCCTG
  • 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 cspA structural gene can be removed and be replaced by a foreign gene.
  • the inverted repeat at the 3' end at 857-866 and 869-878 may be conserved; but if not, the Hindlll fragment would not need to contain the base pairs upstream of the TAA stop codon.
  • the foreign gene (or part thereof) would be inducible by the cold-induced promoter (or its equivalent) and be capable of encoding a target protein.
  • the cold-shock protein would be expressed by the gene coding for it under the control of the promoter of the invention.
  • a promoter other than the native promoter can regulate expression of the cs7.4 gene at physiological temperatures, i.e. , within the temperatures range at which bacteria exponentially grow.
  • a heterologous promoter an E_ ⁇ coli lac promoter, has been used to regulate the expression of the cs7.4 gene.
  • the cspA structural gene was subcloned into a high level expression vector, pINIII (lpp ⁇ ) (7) .
  • the resulting construct, pJJG12 is schematically shown in FIG 8.
  • IPTG isopropylthiogalactoside
  • 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.
  • a second plasmid, pJJG08 (see FIG 9) , was constructed which contains a smaller nucleotide fraction of the upstream region of the cspA gene, terminating at the ApaLI site (bp 534) .
  • the results are shown in Table I.
  • the difference in yields between pJJG04 and pJJG05 would tend to suggest that the promoter or other regulating elements are in the 0 to 534 base pair fragment; the region from 534 to the start of the gene may also embody regulatory elements . Likewise , the region downstream of the gene to bp 878 may also embody regulatory elements.
  • the results show that the cspA promoter is capable of directing a heterologous gene to express a selected protein.
  • the resultant autoradiogram indicated a protein of 8 kdal apparent molecular weight produced only after shift to 25° C . No corresponding band was seen in the pre-shift or 43° C shifted cultures. This protein is designated cs7.4.
  • 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:
  • chromosomal DNA was prepared from overnight cultures of E_ ⁇ coli SB 221 (7) .
  • the cells were collected by centrifugation and washed with lOmM Tris-HCl (pH 8) and lysozyme in 0.25M Tris-HCl (pH 8) was added to a concentration of 3.3 mg/ml.
  • the sample was then incubated at 0°C for 20 minutes followed by the addition of RNaseA to a concentration of 60mM. After 5 more minutes on ice 10% SDS was added to a concentration of approximately 1%.
  • the sample was then mixed rapidly, and the same volume of RNaseA that was added previously was again added along with pronase in 25mM Tris-HCl (pH 8) to a concentration of 0.1 mg/ml.
  • the sample was then incubated at 37°C for 30 minutes followed by phenol extraction, chloroform-isoamyl alcohol (24:1) extraction, and ethanol precipitation.
  • the sample was then further subjected to phenol extraction, ether extractions, and ethanol precipitation.
  • Chromosomal DNA was subjected to drop dialysis using Millipore Type VS 0.025 um filter before restriction enzyme digestion. Chromosomal DNA fractionation was carried out by digesting at least 50 ug DNA with Hindlll, Sail, BamHI, Pstl, and EcoRl and electrophoresing on a 0.7% agarose gel.
  • 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.
  • pUC9 plasmid DNA was digested with Hindlll and ligated with fraction 7 of the Hindlll chromosomal digest (see Example 2) using T4 DNA ligase.
  • E ⁇ coli strain JM83 ara A ⁇ (lac-proAB) rpsL 80 lacM15
  • the probe used was the same one used for Southern blot analysis (see Example 2) .
  • the hybridization temperature was 32° C .
  • a colony which lighted up upon autoradiography was subjected to a second screening by colony hybridization to ensure that the clone had been obtained.
  • 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.
  • This fragment was then ligated with T4 DNA ligase into the promoter-proving vector (pKM005 (Masui et al) (21) after treatment of the vector fragment with Xbal restriction enzyme and Klenow fragment of DNA polymerase I.
  • the E ⁇ coli lac deletion strain SB4288 (21) was transformed and cells carrying the recombinant plasmid were selected as blue colonies on L-agar plates containing 50 ug/ml ampicillin and 40 mg/ml Xgal.
  • the cspA structural gene was subcloned into a hig . level expression vector, pINIII (lpp 3 ⁇ ) (23) using an Xbal site created just upstream of the structural gene using oligonucleotide directed site specific mutagenesis .
  • expression of the cs7.4 protein could be detected by SDS-PAGE analysis of whole cell lysates.
  • Bacteria which are susceptible to transformation include members of the Enterobacteriaceae , such as E-_ coli, Salmonella; Bacillaceae, such as subtilis , Pneumococcus ; Streptococcus ; yeasts strains and others.
  • 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.

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.
PCT/US1990/000776 1989-02-13 1990-02-13 Proteine de recombinaison protegeant du choc du froid, sa production et son utilisation dans l'agriculture WO1990009447A1 (fr)

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Cited By (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)
EP0966541A1 (fr) * 1996-12-19 1999-12-29 University of Medicine and Dentistry of New Jersey Procede et produits de recombinaison pour inhiber l'expression des proteines dans des bacteries
EP1033408A1 (fr) * 1997-11-20 2000-09-06 Takara Shuzo Co, Ltd. Vecteur d'expression inductible a froid
EP1452596A1 (fr) * 2001-11-19 2004-09-01 Riken Promoteurs reagissant aux contraintes de l'environnement et genes codant pour le facteur de transcription
WO2005033318A2 (fr) 2003-09-29 2005-04-14 Monsanto Technology Llc Procedes d'amelioration de tolerance au stress sur les plantes et compositions correspondantes
CN1886514B (zh) * 2003-09-29 2015-11-25 孟山都技术有限公司 用于增强植物胁迫耐受性的方法
US9422599B2 (en) 2008-02-29 2016-08-23 Rutgers, The State University Of New Jersey Cold shock protein compositions and methods and kits for the use thereof
CN110467655A (zh) * 2019-08-14 2019-11-19 上海交通大学 一种蛋白质及其应用

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WO1983003831A1 (fr) * 1982-04-23 1983-11-10 The Regents Of The University Of California Nouveaux micro-organismes de formation de germes de cristaux de glace

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WO1983003831A1 (fr) * 1982-04-23 1983-11-10 The Regents Of The University Of California Nouveaux micro-organismes de formation de germes de cristaux de glace

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Proc. Natl. Acad. Sci. USA, Volume 77, No. 1, January 1980, T. HORII et al.: "Organization of the recA Gene of Escherichia Coli", pages 313-317 *
Proc. Natl. Acad. Sci. USA, Volume 87, January 1990, J. GOLDSTEIN et al.: "Major Cold Shock Protein of Escherichia Coli", pages 283-287 *

Cited By (23)

* 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)
EP0966541A1 (fr) * 1996-12-19 1999-12-29 University of Medicine and Dentistry of New Jersey Procede et produits de recombinaison pour inhiber l'expression des proteines dans des bacteries
EP0966541A4 (fr) * 1996-12-19 2005-04-20 Univ New Jersey Med Procede et produits de recombinaison pour inhiber l'expression des proteines dans des bacteries
EP1033408A1 (fr) * 1997-11-20 2000-09-06 Takara Shuzo Co, Ltd. Vecteur d'expression inductible a froid
EP1033408A4 (fr) * 1997-11-20 2004-08-25 Takara Bio Inc Vecteur d'expression inductible a froid
US6897042B2 (en) 1997-11-20 2005-05-24 Takara Bio, Inc. Low-temperature inducible expression vector
CN100359012C (zh) * 2001-11-19 2008-01-02 独立行政法人理化学研究所 环境胁迫应对启动子和编码环境胁迫应对转录因子的基因
EP1452596A1 (fr) * 2001-11-19 2004-09-01 Riken Promoteurs reagissant aux contraintes de l'environnement et genes codant pour le facteur de transcription
US7368630B2 (en) 2001-11-19 2008-05-06 Riken Environmental stress-responsive promoter and a gene encoding environmental stress-responsive transcriptional factor
EP1452596A4 (fr) * 2001-11-19 2005-10-12 Riken Promoteurs reagissant aux contraintes de l'environnement et genes codant pour le facteur de transcription
WO2005033318A2 (fr) 2003-09-29 2005-04-14 Monsanto Technology Llc Procedes d'amelioration de tolerance au stress sur les plantes et compositions correspondantes
WO2005033318A3 (fr) * 2003-09-29 2005-08-04 Monsanto Technology Llc Procedes d'amelioration de tolerance au stress sur les plantes et compositions correspondantes
KR100851686B1 (ko) * 2003-09-29 2008-08-11 몬산토 테크놀로지 엘엘씨 식물에서 스트레스 내성을 향상시키는 방법 및 이들의 방법
US7786353B2 (en) 2003-09-29 2010-08-31 Monsanto Technology Llc Methods for enhancing drought tolerance in plants and compositions thereof
EP2281895A3 (fr) * 2003-09-29 2011-04-20 Monsanto Technology, LLC Procedes d'amelioration de tolerance au stress sur les plantes et compositions correspondantes
EP2371841A1 (fr) * 2003-09-29 2011-10-05 Monsanto Technology LLC Procédés pour améliorer la tolérance au stress dans les plantes et compositions correspondantes
AP2697A (en) * 2003-09-29 2013-07-17 Monsanto Technology Llc Methods for enhancing stress tolerance in plants and methods thereof.
CN103589749A (zh) * 2003-09-29 2014-02-19 孟山都技术有限公司 用于增强植物胁迫耐受性的方法
US9068195B2 (en) 2003-09-29 2015-06-30 Monsanto Technology Llc Methods for enhancing stress tolerance in plants and compositions thereof
CN1886514B (zh) * 2003-09-29 2015-11-25 孟山都技术有限公司 用于增强植物胁迫耐受性的方法
CN103589749B (zh) * 2003-09-29 2018-11-02 孟山都技术有限公司 用于增强植物胁迫耐受性的方法
US9422599B2 (en) 2008-02-29 2016-08-23 Rutgers, The State University Of New Jersey Cold shock protein compositions and methods and kits for the use thereof
CN110467655A (zh) * 2019-08-14 2019-11-19 上海交通大学 一种蛋白质及其应用

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JPH04500459A (ja) 1992-01-30
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EP0423264A1 (fr) 1991-04-24

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