JPH04500459A - Recombinant cold shock proteins, their production and use in agriculture - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Recombinant cold shock proteins, their production and use in agriculture Industrial applications TECHNICAL FIELD The present invention relates generally to the field of biotechnology, and more particularly to the field of biotechnology. It is now known that cells such as cells can be protected from the negative effects and damage caused by low temperatures. believed to be a novel beneficial drug that is stable at physiological temperatures, based on data from related to a protein (antifreeze protein). The present invention also provides this protein Relating to a method of manufacturing. Furthermore, the present invention provides that this protein or heterologous (he A novel method that regulates the expression of genes encoding terologous proteins. Regarding promoters. Furthermore, the present invention provides that the expression is driven by a heterologous promoter. Cold syrup structural genes that can be regulated by Furthermore, the present invention provides A transformed competent host, a transgenic that is capable of expressing antifreeze proteins. for genetic plants and some other useful applications in agriculture and other fields. related.

Background of the invention 1 Technical field Damage to crops due to frost is due to the loss of agricultural production due to the autothermal phenomenon of weather changes around the world. This is one of the main causes of loss.

It is estimated that 5-15% of the world's total agricultural production is lost each year due to frost damage. It is. In some regions, losses may reach 100%. in the US Economic losses to crops due to frost damage are reported to exceed $5 billion annually.

Most of this damage is caused by syringae scoronaraclens 5pi. Pseudoao such as si%tabacl or r　I　uoreseens Xanthomonas species such as nas species, trans Iucens, etc. or natural ice-nucleated bacteria, such as Ervinla species such as Herblcola. (Ice-nucleating bacteria) caused by a certain type of bacteria. caused by knots. Such natural epiphytic Cepl on the plant surface phytIc) The presence of bacteria causes the nuclei of ice crystals to form at temperatures slightly below 0°C. Can promote formation and formation. It has such ice nucleation ability (IN A”) Bacteria can be found in corn, soybeans, wheat, tomatoes, pears, almonds, and apples. , delicious fruit trees such as cherries and many subspecies such as citrus fruits and avocados. Frost causes frost damage to many important agricultural crops, including tropical plants.

Removal of ice-nucleating bacteria from frost-sensitive plants has immediate and obvious economic benefits.

Due to this need, many technology was born. One technique is to remove the ice nucleation gene from bacteria and Re-introducing these genetically engineered microorganisms (OEM) into crops. Based on.

When these OEMs colonize plants, they replace natural ice-nucleating bacteria. , thereby providing a method of protection against frost. I would like to try to solve this problem Some patents will be discussed later. One of the important aspects of the present invention is , which suggests an approach to solving this problem. Therefore, the US and and provide ways to limit damage to plants and other biological materials worldwide. There is an urgent economic imperative to do so. Treatment of plants with bacterial strains is widely used in the United States and and other matters of legal and public concern, the present invention, in its important aspects, In one, we suggest an alternative to the application of non-ice-nucleated bacterial strains. The present invention Based on the evidence available to date, as described in more detail below, antifreeze Provides a protein that is believed to function as a binding protein. Moreover, the present invention , by integrating a DNA sequence carrying the gene into a recipient crop host cell. Plants that synthesize light anti-freeze proteins or confer similar anti-freeze properties Transforming the plant to make it less sensitive to or resistant to cold temperatures It is also intended that

There are still other important areas to which the present invention pertains. to recombinant DNA or fermentation techniques. The proteins thus produced are enzymatically degraded at physiological temperatures and their The result is modification, reduction, or destruction of the protein's desired biological activity. Examples are known. Furthermore, the normal growth temperature of E. coli (37°C) Misfolding can occur in proteins that are that a complete or partial loss of the desired property (e.g. biological activity) will occur. There is recent evidence to show that. In another important aspect, the present invention provides We demonstrate a solution to these problems by producing the protein at temperatures below the normal growth temperature. It seems to be a good thing.

Therefore, genes encoding commercially valuable proteins can be grown at normal growth temperatures. that the protein can be regulated to be produced only after the temperature has been lowered below By controlling the expression of the target protein to the desired physiological activity of the protein. It can proceed at optimal temperatures without adverse effects.

2 Background technology A fairly extensive literature (including US and foreign) has been published regarding the field of genetic engineering. has been expressed. Where deemed appropriate to aid the understanding of those skilled in the art, Such documents are cited for reference in the description or in the appendix. many patents and references are listed as "References" in the Bundle of this specification.

All such references are hereby incorporated herein.

U.S. Pat. No. 4,766,077 (19811) describes ice nucleation activity (IN A) Within the genomic DNA sequence (INA gene) encoding the polypeptide responsible for Concerning ice nucleation deficient (INA-) microorganisms in which irreversible mutations have been induced. be. The disclosed INA microorganisms are from INA microorganisms forming plant colonies. Applied to plant parts before being established. The process according to this patent All or part of the genes involved in ice nuclei in Toro are subject to notification of missing applications. That is clear.

U.S. Pat. No. 4,375,734 (1983) also states that using a non-phytotoxic virulent bacteriophage that exhibits species specificity for ice-nucleating bacteria. Another ice nucleation inhibitor, Erhl.

U.S. Pat. No. 4,464,473 (1984) describes D Provides a method for isolating a DNA fragment, which DNA fragment is then isolated in a suitable vector. introduced into the vector.

Ice-nucleating microorganisms can be used to prevent supercooling of water in a variety of commercial applications.

U.S. Patent No. 4,161,084 (1979) discloses that plants are To provide a method of lowering the temperature at which freezing occurs. This occurs before freezing temperatures are reached. and, preferably, adding non-ice-nucleating bacteria to the plant at the seedling stage of the plant. achieved by.

Treatment of plants with Ervjnla herbicola before freezing temperature The earlier U.S. Pat. No. 4.045,910 (1977) described also referenced.

If we look at some of the US patent documents in this way, we can see that the problem of frost damage to agricultural crops is not a problem. It is well recognized and many proposals have been made to alleviate this serious problem. I can see what has happened. However, none of these patents teach or teach the subject matter of the present invention. is not meant to suggest.

A review of the technical literature can be found in the following publications:

Proteins synthesized in microorganisms by raising the temperature of the growing culture, by lowering the growth temperature. related to proteins synthesized by cold shock, so-called “cold shock” proteins, Related publications are receiving particular attention.

Where a complete citation is not provided herein, numbers in parentheses indicate References are made to the bibliography in the booklet section.

Ne1dhart, F., C. et al. Regulation (The Genetlcs and Regulaton of Hea) t-Shock Protelns) J, Ann, Rev, Gene t, + Vat.

18, pp. 295-329, 1984) in prokaryotic and eukaryotic cells. The heat shock response is discussed. Heat shock identified in E. coli Proteins are described.

Covlfng% D, W, et al. Consensus 5equencefor Escher ichla colI Heat 5hock Gene Promoters ) J, Proc, Natl, Acad, Sc1. , Vat, Ii2 , I)p, 2679-2683.1985) is an E. coli heat shock protein. Genes encoding motor consensus sequences and heat shock proteins The child has been identified. Transcription from each promoter is heat-inducible in vivo. Therefore, the RNA polypeptide containing the σ70 factor encoded by rpoH (htpR) RN recognized in vitro by merase but containing the major E. coli σ30 factor Not recognized by A polymerase.

The induction of transcription of heat shock proteins is mainly caused by rpoH (HtpR). is achieved by a separate σ subunit of RNA polymerase encoded by It has been reported (Grossman et al. (1) and Trauss et al. (2) )).

For a review of heat shock responses in various organisms, see Lindquist (r Heat 517 response (Heat 5hockResponse) J , Ann, Rev. Biochem, Vol. 55. pp.

151-1101, 198G) and Neldhardt et al. Protein Genetics and Regulation (TlIB gH6ties and Regul ation or Heat 5hock Protelns) J, Ann, Rev.

Genet, Vol. 18, pp. 295-329, 1984) Please refer.

Broeze et al. (24) showed that E. coli and P, fl after a temperature shift to 5°C. have reported differences in protein synthesis by S. urascens. Mesophilic (me) In E. coli (sophilic), protein synthesis declines for 1 hour and then stops. It was reported that the 70s Liposo found after such a temperature shift The accumulation of mucosal proteins has been interpreted as indicating inhibition of translation initiation. On the other hand, the shift to 10℃ The result is a 4 hour growth delay followed by new growth. Ng et al. (4) and Jones et al., supra.

For further background on anti-freeze proteins, see ) few et al. (5) and D. See uan et al. (6). Such proteins are found in polar oceans. It is common in high concentrations in the serum of western fish and in the hemolymph of insects in areas where the temperature is below freezing in winter. It is a low molecular weight protein found in

For background information related generally to the regulation of transcription, see, e.g. Protein-nucleic acid interaction: Molecular analysis (Protein-Nuclelc Ac1d 　Interaetfons　1nTranscript1on　:　A　Mo 1ecular Analysis) J, Hlppel.

P, H, et al: Ann, Rev, Blochem, Vol, 53. pp. 389-440.1984). Others related to this description For publications that can be referred to, please refer to the bibliography of Tadabe. In this specification Some patents in the field of genetic engineering that use terminology and techniques used in ``Novel cloning for the expression of polypeptides in microbial hosts'' Carrier CNove! Clonlng Vehlcles for Polypept lde Expression) n Mlcroblal 1 asts) J and U.S. Pat. No. 4,824,928 entitled e and Kenzo Nakamura, 19116), [in microbial hosts A novel cloning vehicle for the expression of polypeptides lng Vehlcles ror PolypeptjdeExpressi U.S. Patent No. 4.68 entitled on tn Microblal Ho5ts) J. No. 6,838 (Masayorf Inouye and Kenz.

Nakamura, 1987), “Expression of polypeptides in microbial hosts. A novel cloning carrier for s for Polypeptide Expression 1n)llcr U.S. Pat. No. 4,843,989 entitled Masayorl 1 nouye and Yoshihlro Masul, 19 87), and [Clonin for expression of polypeptides in microbial hosts. Ctoning carriers for Polypeptide Expression In Mlcroblal Ho5ts) entitled J. U.S. Patent No. 4,757. Specification No. 13 (Masayorl 1 nouye and and Yoshlhlro Masul, 19811).

Other publications of interest include those published recently (“Low temperature in Escherichia coli”). Induction or Proteins in Re5ponse to Low Temperatures 1n E xcherichia colt) J, Jones, P, G.

Ra: The Journal! orBacteriology IVol, 189, pp.

2092-2095, 191:l. In this document, transcription and translation or oral 1 The synthesis of a group of proteins involved in RNA degradation has been discussed.

When the bacterial growth temperature is lowered from 37°C to 10°C, the protein retards growth. It has been reported that it is synthesized during About 13 proteins were mentioned ing. Twelve identified proteins were synthesized during growth lag (temperature increased from 37°C to 5 have not been identified and are one of the cold shock proteins. (referred to as Flo, 6) is detectably synthesized during growth at low temperatures. . There is no indication that this is a temporary synthesis.

Brief description of the drawing Figure 1 shows the induction of major cold shock proteins. The arrow to the right indicates The major cold shock protein, es7.4, is shown.

Figure 2 (A and B) shows the temporal induction of cs7.4. The number shown is below It is as follows. 1) Pulse-label, 0-30 minutes after temperature shift, 2) 30-6 0 minutes, 3) 60-90 minutes, 4) 90-120 minutes, 5) 120-150 minutes, 6) 1 50-180 minutes. The arrows are as shown in FIG.

Figure 3 shows the stability of cs7.4. The arrow indicates cs7.4.

The fourth column shows the two-dimensional gel used in the purification of cs7.4.

Figure 5 shows Southern blot analysis of cspA.

Figure 6A shows the DNA sequence of cspA (restriction map and sequencing procedure).

Figure 6B shows the partial nucleotide sequence of the cloned Hindm fragment. . The amino acid sequence corresponding to the espA coding region and cs7.4 is shown in bold. show.

The underlined AGG is probably Shine-Delgarno. arno) array. Regions underlined with half-arrows indicate inverted repeats.

Figure 7 shows plasmid pJJGO1 carrying the cspA gene (indicated by an arrow). This is what is shown.

FIG. 8 illustrates pJJG12.

FIG. 9 illustrates pJJGO4.

Summary of invention disclosure The present invention involves genetically transforming microorganisms, especially bacteria, and imparting them with genetic capacity. especially those that do not produce "cold shock" or "anti-freeze" and other proteins. Methods, systems, and compositions are provided. Also, RNA polymerase Correct binding of the holoenzyme and subsequent conversion into a form capable of specific RNA transcription initiation A DNA fragment (a "promoter") containing a signal that directs activation is provided.

The promoters described herein are inducible by cold and call Expression of the cspA gene, which encodes shock or antifreeze or other proteins. You can adjust the current state. The specific application is as shown below. Ru.

Proteins called cold shock or antifreeze proteins, especially cs7. Genetics for expressing the E. coli cold shock protein, called 4. system is provided.

In response to a decrease in temperature below ambient temperature or below physiological growth temperature, the present invention provides: Novel polypeptides synthesized in E. coli are provided. polypeptide (also is a 7.4 kdat protein induced under cold shock. It is called rcs7.4J due to its quality.

What is remarkable about this polypeptide is that it is It must be stable at temperature.

The present invention also provides a gene encoding cs7.4 protein. moreover , the present invention regulates the expression of the gene encoding cs7.4, and also inhibits the growth of microorganisms. Transcription of proteins other than cs7.4 is initiated in response to a temperature shift below the growth temperature. provides a cold-inducible promoter that can be

The present invention also provides antifreeze protection under the direction of a promoter other than the promoter of the present invention. A system for expressing genes encoding proteins is provided.

Additionally, the invention provides promoters, structural genes and other necessary functional DNA elements. Various DNA constructs such as plasmids and loning vectors containing and providing a transformed host.

The present invention also provides protection against cold-induced injury or lethality, such as freezing of microorganisms and plant cells. The products of the present invention can be used in various ways in the field of preventing or reducing dryness, etc. It is also intended. The proteins of the invention can be used as "antifreeze" compounds for crops, for example. Also, the gene encoding the cs7.4 protein of the present invention can be used as or use a non-toxic microorganism transformed by a part thereof for agricultural or other uses. It is intended that there be. It also contains a sequence encoding the es7.4 protein. The cs7.4 antifreeze protein is then transferred to crop host cells, which produce the cs7.4 anti-freeze protein. It is also intended to protect crops from frost damage.

The present invention inhibits the expression of cold shock-inducible genes encoding anti-freezing proteins. A cold-shock-inducible promoter (“native”) that can be regulated Promoter).

Additionally, the present invention provides a method for generating genes encoding anti-freeze proteins or other proteins. Promoters that can regulate the expression of ")I will provide a. cs7.4 is often referred to herein, but the It is intended that any protein be produced by the promoter.

The present invention also provides that under the control of a heterologous (exogenous) promoter at physiological temperature Provides for expression of genes encoding antifreeze proteins.

Furthermore, the present invention further clarifies the promoter sequence and provides the D It is contemplated that the NA sequences may be created synthetically. Such promoters , to regulate protein expression at low temperatures, i.e., subphysiological temperatures. It would be useful.

In the present invention, promoters can be used without the native structural gene and vice versa. ``Unlinked'' in the sense that a structural gene can be regulated by a heterologous promoter. (U-coupled) It is noteworthy that it can be J.

Other characteristics of the invention will become apparent from the description below.

Brief description of the drawing (Figure 1 - Induction of major cold shock proteins) Cell culture growing at 30°C The nutrient solution was heated to 42°C, 30°C, 25°C and 18°C, and immediately The cells were pulse-labeled with nin for 10 minutes. Sodium dodecyl sulfate sample The samples were subjected to polyacrylamide gel electrophoresis. Autoradiogram as main image show. Molecular weight standard sizes are shown on the left in kilodaltons. The arrow to the right is guided The major cold shock proteins are shown.

(Figures 2A and 2B - Temporary induction of cs7.4) Cells growing at 37°C The culture solution was divided and kept at 10°C or 15°C. Next, each culture solution was mixed with [35S ] Pulse labeling with methionine at 30 minute intervals and electrophoresis as described in Figure 1. Served. In the autoradiogram shown in Figure 2A, C is the temperature shift. A control is shown in which the aliquots were pulse labeled for 5 minutes at 37°C before testing. The numbers indicate: . Pulse labeling, after temperature shift, 1.0-30 minutes, 2.30-60 minutes, 3.60- 90 minutes, 4.90-120 minutes, 5.120-150 minutes, and 6.150-18 0 minutes. The arrows are the same as described in FIG. Figure 2B: Autoradiogram of Figure A was subjected to scanning densitometry to detect methionine-labeled cs7.4 protein in whole cells. The percentage of protein was determined at each time interval. Each time point on the graph is Shows the percentage of methionine labeled fics7.4 protein at the end of the 0 minute interval. vinegar. For example, between 30 and 60 minutes (time 60 minute point on the ordinate), cs7. 4 accounts for 10% of the total methionine-labeled proteins in the cell at 10°C. 0 hours is , represents a 5 min pulse at 37°C, which is described in Figure A.

(Figure 3 - Stability of cs7.4) Cell cultures grown at 37°C were brought to 15°C.

After 30 minutes, the culture solution was incubated with [35S] Translabel. Pulse labeling was performed for 0 minutes. Next, the culture solution was added to the autoradiogram shown in this figure. non-radioactively labeled methionine and cysteine for various lengths of time as indicated above. It was tracked using Samples were subjected to electrophoresis as described in FIG. 37℃ Samples were prepared as described in FIG. The arrow indicates cs7.4.

(Figure 4 - Two-dimensional gel used for purification of es7.4) Cells growing at 37°C The culture solution was kept at 14°C for 4 hours. Harvest the culture fluid, fractionate it, and collect the cytoplasmic fraction. It was subjected to two-dimensional gel electrophoresis. The first dimension is isoelectric focusing, the second dimension is 5DS - Polyacrylamide gel electrophoresis was performed. Electrate the gel onto the PVDF membrane. Loplotsi, stained with Coomassie blue dye. The arrow indicates CS7.4.

(Figure 5 - Southern blot analysis of cspA) Escherichia coli chromosomal DNA was collected using various restriction methods. After enzymatic digestion, the DNA was transferred from the agarose gel to nitrocellulose paper. Ta. Hybridization using the degenerate probes described herein. Dization was performed to obtain the autoradiogram shown in this figure. restriction enzyme digestion are S (Sall), P (PstI), B (BamHI), E (Ec oRI) and H (HindIff).

λ DNA was digested with HindIn and used as a size standard. HindI Chromosomal DNA digested with ll was also fractionated by agarose gel electrophoresis and shown in this figure. The indicated fractions 1-12 were obtained.

(Figures 6A and 6B - DNA sequence of cspA) 2 from clone pJJGO1 ．． Approximately half of the 4 kb HindII fragment was sequenced. Figure 6A shows restriction enzymes The map and sequencing process are shown. The restriction enzyme site is H(HfndI [[), D (Dral), S (Smal), B (BglI), A (ApaLri, P (Pv uII) and X (XmnI). The thick arrow indicates the region encoding cspA. show. Figure 6B shows partial nucleotide sequence of the cloned HindIII fragment. The code array is shown. Corresponds to the region encoding cspA and cs7.4 Amino acid sequences are shown in bold. Underlined AGG is the expected SD arrangement It is. Regions underlined with half-arrows indicate inverted repeats.

Factors 7.8 and 9 are further described below.

Description of specific and preferred embodiments The present invention provides a method for producing cold shock J protein, a promoter therefor, and a method for producing cold shock J protein. and various constructs.

In one embodiment described below, the protein encoding the cold ship protein The gene is expressed under the control of a heterologous promoter. In other embodiments, the code Rudoshock-induced promoters exhibit differential activity in response to reduced growth temperatures. Used to regulate the synthesis of seed proteins.

The method of the present invention for inducing and producing the cs7.4 protein of the present invention involves microorganisms, e.g. E. coli, at an exponential rate in a high nutrient medium at the physiological growth temperature of each microorganism. It consists of growing to the desired growth density. In E. coli, such a temperature is It ranges from about 0°C to about 50°C, preferably from 20°C to about 40°C. microorganisms Each has its own optimal growth temperature. For E. coli, raise the temperature to approximately 40°C. When the temperature is lowered to below 20°C, growth gradually slows down, and the upper limit of the growth temperature, approximately 49°C, Or at the lower limit, about 8°C, growth stops.

When the desired degree of growth has been achieved (monitored by a suitable method such as spectrophotometry) temperature of about 10°C or higher and about 20°C or lower, preferably about 20°C or lower. and then rapidly shift to a lower temperature of about 8°C or higher. In general, at low temperatures, the growth rate is not maintained. If desired, at a low temperature but without the growth of microorganisms. The temperature may be shifted to a higher temperature. The present invention can be applied for a suitable period of time in a low temperature range. for optimal production of polypeptides. The kinetics of polypeptide induction is Pulse label with radioactive methionine, collect cultures and subject to two-dimensional gel electrophoresis. Therefore, the amount of protein synthesized by processing and separation and autoradiography tracked by any suitable method, such as by determining the

The proteins of the present invention are suitable for physiological growth in which a microorganism, in this case E. coli, grows normally. This polypeptide can be synthesized at lower temperature ranges. was found. Rapid induction of polypeptide synthesis is induced by heating to 10°C or 15°C. It usually happens during the first 30 minutes after a shift. Maximum induction and rate of synthesis is temperature Depends on temperature after shift. After the shift to 15°C, is the maximum synthesis due to a temperature shift? is achieved after 30 to 60 minutes, and the maximum synthesis rate is approximately 13.1% of total protein synthesis. %. See 2A and 2BC. If shifted to 10℃, shift The highest synthesis rate of about 8.5% of total protein synthesis occurs 60 to 90 minutes after injection. However, Therefore, adjustment of the temperature can be adapted to the purpose of the present invention to improve the polypeptide synthesis rate and/or or allow for yield adjustment. After maximum total polypeptide yield is achieved, The rate of polypeptide synthesis decreases and reaches a maximum in cultures temperature-shifted to, for example, 15°C. approximately one-fifth of the time, and approximately three-fifths of the maximum for cultures temperature-shifted to 10°C. will eventually be reduced.

A notable feature of the cs7.4 protein of the present invention is the temperature range in which it is induced and synthesized. stability at temperatures higher than the ambient temperature. Such physiological temperatures range from about 5°C to about in the range of 40°C or higher. The data in Figure 3 shows that proteins are synthesized. It was stable for 20 hours at 15°C (only about 30% of the protein was degraded), and 3 It has been shown to be stable at 7°C (for at least 1.5 hours).

The stability of the proteins of the invention at physiological temperatures is important for practical applications.

This allows physiological temperature control using promoters other than the promoters of the present invention. The synthesis of this protein is possible. Additionally, this protein may be frozen or frost damaged. Apply the protein agriculturally to crops at ambient temperature before initiating its preventive function. It becomes a product.

Other elements of the invention are described below. The promoter of the present invention The cloned HindII [located between nucleotides 1 and 605 of the fragment] It is believed that there are. The first 997 bp of the cloned Bindu fragment contains all essential requirements of a functional gene for regulated expression, including liposome binding sites. Contains elements.

one 35 upstream and one 10 upstream of the coating region, i.e. 330 positions each and proof of promoter sequence at position 355. Other characteristics of promoters is to respond to a drop in temperature.

The promoters of the invention can be activated at reduced temperatures to promote transcription of the genes of the invention. Instruct the photo.

The promoters of the present invention are cold inducible in vivo and are able to induce RNA polypeptides in vivo. Recognized by merase.

We do not subscribe to any particular theory or principle regarding mechanism of action or function. I don't want to be fooled, but several hypotheses are currently being considered. The inventors There are three possibilities (overlapping possibilities) as the mechanism of regulation of the promoter of the present invention. I am thinking that there is a possibility. First, the inducer activated by low temperature There is a possibility that expression from the promoter can be induced at low temperatures. Secondly, Cold-sensitive repressors are inactivated at low temperatures, resulting in gene expression. Maybe it will be done. Third, the upstream of the structural gene by RNA polymerase A cold-inducible alternative (such as σ70) that allows recognition of novel promoter sequences It is conceivable that genes are regulated by sigma factors (other than standards). Special The host transformed into Enterobacteriaceae (Enterobacteriaeeae) Another mechanism may be postulated, considering that it may not belong to

The present invention further provides cs7. 4 is provided. This polypeptide partial amino acid sequence, SGKMTG (X)VKWFNADKGFGF I. do. Here, X is leucine or isoleucine. isoleucine and leucine Both syns were identified (64% and 36%, respectively). Therefore, the present invention and both polypeptides. The polypeptide of the present invention has 70 amino acids. It is a protein consisting of acid residues. The calculated molecular weight is 7402 Daltons. , the calculated pI is 5.92. Polypeptide contains 20% or more charged residues It is extremely hydrophilic.

Lysine residues make up 10% of the protein. According to the NBRF database, other No homology with any sequences was observed.

Chou and Pa According to the rule of sman (1978) (11), remarkable secondary structure forms are not recognized. There wasn't. However, amino acid residues are put into a helical net. When lotted, 12 of the 16 charged residues are adjacent as oppositely charged pairs. It was decided that. Also, when a protein is folded into an α-helix, 12 of the 16 charged amino acid residues in a protein are oppositely charged residues It becomes parallel. This data indicates that most or substantially all of the proteins of the invention This suggests that the part may have an α-helical structure.

Secondary structure of antifreeze polypeptide from fish (Winter Flounder) Recent crystallographic studies show that the molecule is a single α-helix . Crystal structure of antifreeze polypeptide and its mechanism of action (CrystalS structure of an Antifreeze Polypeptide e and its Mechanistic Implications. Y ang et al.: Nature. Vol.

333, 19 May 1988, pp. 232-237) (25) Please refer. The mechanism of antifreeze protein binding is that the entire surface of the ice crystal is a protein surface. Water It is proposed based on the fact that many patterns of disjoint bonds can exist. Show The suggested mechanism is that the antifreeze polypeptide induces local alignment of the ice lattice, resulting in a helical structure. The dipole moment from directs the preferred alignment of the peptide to the C-axis of the ice nucleus, and then A shift in the helical structure can occur due to twisting movement of the side chain of the hydrophilic amino acid. The idea is that the bond between the protein and the ice surface is strengthened.

Further demonstration of the helical structure of the polypeptides of the present invention provides that the polypeptides of the present invention cs7.4 can be produced by genetic engineering methods in E. coli at low temperatures. It remains to be seen whether this is the first anti-freeze protein to be induced. cs7. Research on the secondary structure of 4 may lead to other possibilities. For example, α-helix Only those parts of the peptide with structure are essential for antifreeze function, and similarly, such Only the portion of the nucleotide sequence encoding the specific peptide fraction is suitable for such cryoprotection. It can be assumed that it is essential for

Cloned 2,4kb HindII containing gene for cs7.4 I fragments were digested with Hindm and separated on a 5% polyacrylamide gel. isolated from pUC9 by. The fragment was then subcloned into M13. Ta. DNA sequencing was performed using the chain terminus method (Sanger et al.: 197 7). DNA sequencing was performed using [35S]dATP and enzymes, Manufacturer (υnited Stat) using Sequenase esBlocheslcal Corporation). went.

The partial nucleotide sequence of the cloned HindI[I fragment is cs7. 4 and its promoter. Polypeptide cs of the present invention The nucleotide sequence encoding pA contains the following 210 nucleotide sequence: nothing.

AT” CrCCTGAC GA n ass is αkoAαπ CTG The corresponding amino acid sequence encoded by cspA is shown below.

H@ts@rGlyLyd*tzyX1mVa31. ysTrp Ph mountain hunger rabbit LYsGlyPi+eGlyPhe 工1eτhrPr.

StatusμU GlysarLysAspvau’b@ValEisPbe5框HK社menC RinAspGLy? yrLysS@rLauAspGluGlyGj≦Y薯Vt LSarPh*SynsGlusarcLyAlaLynyb-collapseGlyAsn Val? The hrSarLmu sequence is shown in Figure 6B and was cloned. HindI [[TAA starting from the ATG codon at nucleotide 61 of the fragment Oven reading frame consisting of 210 nucleotides ending with a stop codon It includes an open reading frame. This oven ready The coding frame is derived from the gene herein named cspA, which is involved in cs7.4 synthesis. This is the child coding area.

The present invention has within its scope the polypeptide cs7.4 or the properties of cs7.4 a nucleotide sequence encoding a polypeptide (functionally equivalent) or It also includes any subsequence. The present invention also provides that one or more codons If the nucleotide sequence is replaced by some other codon, the cs7.4 point Any equivalent that encodes a repeptide or is functionally equivalent to it. Also includes nucleotide sequences.

In studies related to the present invention, encoding the cold-inducible polypeptide cs7.4 Two copies of the gene (espA and cspB) may exist Evidence has been presented to date to suggest this. More on this evidence below think about. The present invention includes within its scope cs7.4 polypeptides or functional equivalents thereof. and other possible genes encoding equivalents. In molecular biology, Examples of multiple genes encoding the same protein in E. coli, e.g. -Sidin factors Tu (tufA and tufB) and ornitinka Rubamoyltransferase (Arg ArgFArgl) and the like. cs Given that there are two genes encoding 7.4, this is a two-gene cold-inducible polypeptide. This was the first discovery of a peptide.

According to the present invention, the 997 bp fragment of the cloned HindIII fragment It is conceivable that the cspA structural gene could be removed and replaced with a foreign gene. It will be done. If necessary, reverse the 3' ends of 857-866 and 869-878. may hold repetitions of . However, if not needed, the HindI[I fragment is TA There is no need to include base pairs upstream of the A termination codon. Exogenous gene (or part thereof) minute) is induced by a cold-inducible promoter (or equivalent) can be obtained and encode the protein of interest.

In other embodiments of the invention, the cold shock protein is a gene encoding the cold shock protein. The gene is expressed under the control of the promoter of the present invention.

In yet another aspect of the invention, the promoter other than the original promoter is es7.4 genes at physiological temperatures, that is, the temperature range where bacteria grow exponentially. Gene expression can be regulated. Therefore, according to the present invention, heterogeneous promotion The E. coli 1ac promoter regulates the expression of the cs7.4 gene. It was used in

The cspA structural gene was transformed into a high-level expression vector, pIN■ (Ippp5) (7 ), was subcloned into. The resulting construct, pJJG12, is illustrated in Figure 8. As shown. Add isopropylthiogalactoside (IPTG), Expression of cs7.4 protein at 7°C was determined using SDS polyacrylate in whole cell lysates Detected by amide gel electrophoresis. Protein expression is dependent on the native promoter. 5 to 0 times less than that obtained from expression controlled at 15°C by a It was.

However, the expression of this protein is caused by a promoter other than the original promoter. The fact obtained at higher temperatures is especially important since this protein is normally expressed at 37°C. This is significant because it does not occur. This opens up many interesting new possibilities. will be held. Promoters other than the lac promoter regulate the expression of this gene. It may also be more effective. In addition, fine particles in the low temperature range (15 to 10°C) The growth of cells is slower than at the optimal growth temperature of the selected microorganism, e.g. E. coli. stomach. Therefore, although yields may be low at optimal growth temperatures, low yields are due to bacterial appears to be covered by faster proliferation of .

Regulation of cs7.4 gene expression using a promoter stronger than the lac promoter If used in It can be expected to be obtained on a commercial scale in time. Therefore, a large amount of antifreeze protein will be obtained.

The above properties, i.e., the protein is produced at physiological temperature and the protein is cryoprotective. Being active as a protein is therefore of great significance.

As a result of this disclosure, those skilled in the art will be able to use other suitable promoters other than the lac promoter. , for example, trp, tac promoter, λpL, ompF Sop p and other promoters, which control the expression of the gene encoding the desired protein. It will be appreciated that it may be used for adjustment.

When expressing proteins using other transformed microorganisms such as yeast, GA LIO and other promoters may be suitable.

Furthermore, as briefly mentioned above, cspA proteins of the invention that are active at low temperatures The motor was stimulated by expression of proteins other than cs7.4 cold shock protein. Can be used for adjustment. This naturally therefore opens up further possibilities. It's a spider. enzymatically (e.g. by proteolysis) at physiological temperatures Proteins, which would otherwise be useful if not degraded, are less susceptible to enzymatic degradation. Of particular interest is that it can be expressed at low temperatures. A similar possibility is , probably due to misfolding when produced at physiologically active temperatures. It is also found in useful proteins that are biologically inactive. of the present invention Produces properly folded and active proteins under the control of the cspA promoter. It may be advantageous to produce.

These observations not only apply to antifreeze proteins, but also traditionally Any protein that could not be expressed in the desired structural form at the desired temperature It also applies to the expression of texture. These proteins at lower non-injury temperatures It can be expressed using the promoter of the present invention.

In the above aspects of the invention, the cspA promoter of the invention is based on the classical model was used to regulate the expression of β-galactosidase. For this purpose, Plasmid containing the 1acZ structural gene and lacking a promoter (pKMOO5) (21) was inserted into the cspA promoter on the Hindm-Pvun fragment of 806b9. (pJJ'GO4). Please refer to FIG.

A second plasmid, pJ'JGO8 (see Figure 9), was used to carry the cspA gene. Contains a smaller nucleotide fraction of the upstream region of the child, including the ApaLI site (bp53 It was constructed so as to end in 4). The results are shown in Table 1 below.

Table 1 Plasmid temperature 15℃ to 10℃ to 37℃ After shift pKMOO56,74,13,7 pJJGO4549,0900,0g51.0pJJGO840,745,85 The 6,1 numbers represent "units" of enzyme activity.

The difference in yield between pJJGO4 and pJJGO5 may be due to promoter or other regulatory elements. This suggests that it is in a 0-534 bp fragment. From 534 to the start of the gene The region may also contain regulatory elements. Similarly, the remains up to bp878 Downstream regions of the gene may also contain regulatory elements.

The results demonstrate that the cspA promoter has the ability to direct protein expression to the inserted gene. It shows that there is power.

The following examples are merely illustrative of the invention and are not intended to limit the invention. There isn't. Those skilled in the art will readily be able to make changes and substitutions to achieve equivalent results. Ru.

E. coli 5B221 (lpp hsdRtrpE51acY recA/F'1 acIqlac+pro'') (7) was temperature-shifted in a culture volume of 1011. The cells were grown to a density of approximately 2 x 108 cells/-1 prior to cell culture. Grow at 30℃ 1.11 aliquots of the cell culture medium were mixed with 10 uC1 of [353]methionine (^*e rshasCorp,, >1000 C1/m5ol) or [35S]) La ICN Radlochesjcals, IrVIn8, CA ) in beakers at 42°C, 30°C, 25°C and 18°C and incubate for 10 minutes. Russ labeled. All samples were collected by centrifugation and pellets were lyophilized. I let it dry. The sample was then treated with sodium dodecyl sulfate-polyacrylic Applied on a 17.5% separating gel for amide gel electrophoresis (SDS-PAGE) . The gel was dried and exposed to X-ray film.

The obtained autoradiogram shows a protein with an apparent molecular weight of 8 kdal. was produced only after a shift to 25°C. Before shift or to 43℃ No corresponding band was seen in the shifted culture. This protein is c It is called s7.4.

Plus containing sequences encoding cold shock proteins and their regulatory elements In constructing a mido, the first step is to identify and isolate the gene locus (focus). is necessary. To prepare oligonucleotide probes, parts of the protein are Obtain the partial amino-terminal sequence. 101 of the culture solution of E. coli 5B221 (7), Grow at 37°C to a density of approximately 2 x 108 cells/1 and transfer to 14°C for 4 hours. vinegar. Then collect the cells and 4. The soluble fraction was fractionated as described in (9) above. Up Pulse for 30 min after shift to 15°C as described above to remove trace amounts of labeled protein. , then mixed with 250 μg of soluble fraction protein.

Next, two-dimensional electrophoresis was performed, and the -dimension was performed using isoelectric focusing (aspho! 1ne). s, p H3~10.1.5%, pH6~8.0.5%) and the second dimension is 5D S-polyacrylamide gradient gel electrophoresis (10-18.4% acrylamide, degree of crosslinking 2.7%) according to the method of 0° Farrell (15). minutes The released proteins were transferred to a semi-dry block device (Sa+4torius Gmb). )I, Goettlnggen, FRG) and 48mM as transfer buffer) Squirrel, 391M glycine, 1.3gM SDS and 20% (V/V)y1 9) Polyvinylidene fluoride (PVDF) membrane (Mi llipore Corp, Bedford, HA). Ta. The membrane was then protein-stained with Coomassie Blue R-250, dried and , and subjected to autoradiography. previously observed by autoradiogram A darkly labeled protein is clearly visible at a position with approximately the same molecular weight as cs7.4. It was identified as (Figure 4). c on the membrane stained using autoradiogram spots. The s7.4 spot was identified and then cut out from the plot. Automatic App Used Blosysteas Model 470 gas phase sequencer and performed directly on stained membrane fragments. The obtained amino acid sequence is as above. be.

Mixed degenerate oligonucleotide probes for Southern blot analysis are shown below. It was created to match a short region of the amino acid sequence.

,,, put -WF N in +1. + For Southern blot analysis, chromosomal DNA was isolated from E. coli 5B221 (7). Prepared from culture. Cells were collected by centrifugation and 10Il)I Tris-HCl (pH 8) and remove lysozyme (0.25M triz-hydrochloric acid (pH 8) solution). It was added to a concentration of 3.3B/ml. The samples were then incubated at 0°C for 20 min. After incubation, RNase A was added to a concentration of 60×. difference After leaving the mixture on ice for another 5 minutes, 10% SDS was added to a concentration of about 1%.

The sample was then quickly mixed to ensure that an equal volume of the previously added RNase A was added after the addition. Pronase (25℃M Tris) adjusted to a concentration of 0 or 1ag/ml Hydrochloric acid (pH 8) solution) was added again. The sample was then incubated at 37°C for 30 After incubation for minutes, phenol extraction, chloroform-isoamyl alcohol The mixture was subjected to ethanol (24:1) extraction and ethanol precipitation.

The sample was then further subjected to phenol extraction, ether extraction and ethanol precipitation. Served. Before restriction enzyme digestion, chromosome D N A-f-Mllljpore Type VS O,,025u-Fi Drop dialysis was performed using a router.

The chromosomal DNA fraction was analyzed by incubating at least 50 μg of DNA with HindII [, Sal Digested with I, BamHI, PstI and EcoRI and 0.7% agarose This was done by electrophoresis on a gel.

Southern transfer of DNA from agarose gel to nitrocellulose paper was performed using Nan1a The method described in the manual by Tis et al. (19) was performed with the following modifications.

Acid depuration for partial hydrolysis of DNA ) was performed by washing the gel once for 15 min in 0.25 M hydrochloric acid. . Additionally, the transfer pan was filled with 20XSSC instead of 10XSSC.

The hyperdase was prepared using the method of Maniatis et al. (26) with the following modification. I changed it. Prehybridization and hybridization solutions are both 0.1 ml of 50 x Denhardt's, 0, 2 ml of 3 0XNET.

0.51 of 20% dextran sulfate and 0.05 ml of 10% SDS (Izu (per ml of solution).

These solutions are described by Inouye and Inouye (19). P The oligomers used as lobes are shown above. [32P] labeled The probe was created according to Inouye and Inouye (19) and Prehybridization and hybridization were performed at 32°C.

Wash the filter by the method of Nouye and Nouye (19). Cleaned and dried.

Southern blot hybridization silane with mixed oligonucleotide probes Their autoradiogram shows at least one distinct band in each digest. Indicated. In particular, the HindIII digest produced one band with a size of 2.4 kb. (See Figure 5).

A 2.4 kb HindIII fragment was isolated as follows.

The HtndI digest of chromosomal DNA was fractionated on a 0.7% agarose gel.

Next, gel slices were cut out every 0.5 cm from the top surface layer of the gel. Each Gersula The cells were frozen at -20°C for at least 20 minutes and then placed in Eppendorf tubes. Centrifuged for 10 minutes. This was repeated three times, but in the final round, before freezing, Add an appropriate amount of N-Tris and 0.1M EDTA (pH 7.5) and drain the supernatant liquid. Collected after each centrifugation. The sample was then extracted three times with phenol and ether. It was subjected to extraction and ethanol precipitation.

Each fraction was analyzed by Southern blot using probes.

DNA fragments from fractions 7 and 8 represent 2,4k in the original chromosome digest. It clearly hybridized with a probe that corresponds well to the HindIII band of b. .

Example 3 Cloning of cspA gene pUC9 plasmid DNA was digested with HindIII and T4 DNA ligase was added. was used to ligate fraction 7 of the Hindm chromosome digest (see Example 2).

Next, Escherichia coli JM83 strain (ara A (Iac-proAB) rpsL 8 0 IacM15) (13) was transformed with 50 μg/sl ampicillin. 25μl of 40g+g/ml xgal was applied on an agar plate. Cells were screened. Pick up the white colonies on Whatman filter paper,] colonies as described by Nouye and Nouye (19). It was subjected to hybridization screening. The probe used was Southern It was the same as that used for plot analysis (see Example 2). /X Iburi The daidze silane temperature was 32°C. It appeared bright on autoradiography. Colony hybridizer to confirm that clones are obtained. It was subjected to a second screening by ization.

Example 4 Use of the asp promoter to direct heterologous protein synthesis csp promoter in E. coli from plasmid pJJGO4. It was used to direct the synthesis of lactosidase. Construct this plasmid as follows. Ta.

A 2.4 kb HindIII fragment containing the gene was digested with Puvll. obtained Separate the 806 bp fragment on a 0.8% agarose gel and cut out the band. and a salt-bridge electroelution device (IBI * Inc.) according to the manufacturer's instructions. DNA was recovered by electroelution using This fragment was then added to DNAT4 Using ligase, digest the vector fragment with Xba restriction enzyme and DNA polymerase I. After treatment with the Flenow fragment of the promoter-confirmation vector (pKMO05) (Masui et al.) (21). E. coli lac deletion strain 5B4288 ( 21) and transform the cells containing the recombinant plasmid into 50 μg Blue on L-agar plates containing ampicillin and 401 g/ml selected as a colony.

A culture of E. coli 5B4288 containing plasmid pJJGO4 was grown at 37°C. Growth was performed and the temperature was shifted to 10°C or 15°C. β-Garak before and after shift Tosidase activity was determined using the substrate O-nitroph as described by Mutter (22). Determined using phenyl-β-D-galactoside.

The results showed that shifting the culture to 15°C reduced the β-galactosidase activity by 64%. showed an increase in transcription from the cloned cspA promoter and This demonstrates the subsequent induction of β-galactosidase expression.

This example demonstrates the multifunctionality of the promoter sequences of the present invention and the cspA structural gene. In the level expression vector pINItI (lppP5) (23), All structural genes can be synthesized using oligonucleotides directed to position-specific mutagenesis. Subcloning was performed using the Xba1 site created upstream. I PTG's Expression of cs7.4 protein was detected by 5DS-PAGE of whole cell lysates by adding It could be detected by analysis.

Cloning of DNA fragments with promoters and evaluation of promoter effectiveness For vectors that can be used for testing, see Nauf et al. (21). sea bream.

Other competent microbial hosts (eukaryotic and prokaryotic) are subject to this invention. It is understood that it can be transformed directly. Bacteria that are susceptible to transformation include Bacteria of the Enterobacteriaceae family such as Escherichia coli, Salmonella, Bacillus subtilis, etc. using all or part of the synthetic gene or the nucleotide sequence shown in Figure 6. Saccharomyces cerevisi Transformation of yeast cells such as ae) may be of interest. Fundamentals of yeast genetics techniques, suitable yeast cloning and expression vectors and transformation protocols. ``Recent Progress in Molecular Biology'', which is specifically cited herein as a reference. Current Protocols in Molecular Biology) J (Supplement 5, 1989) (23) It is being considered.

Similarly, vertebrate cell cultures may also contain the structural genes of the present invention or portions thereof or may be transformed with all or part of the nucleotide sequence shown in the figure. Not possible. Those skilled in the art will be able to select an appropriate cell culture, such as the CO3-7 system of monkey fibroblasts. will. Suitable techniques for transfection of DNA into eukaryotic cells "Current Protocols) J Chapter 9 ( (also incorporated herein by reference). exemplified The protocol used is HeLa.

Cell lines such as BLAB/c3T3, NIH3T3 and rat fetal fibroblasts Those that can be used with good results are shown.

For further vectors and their sources, please refer to Fermentation with appropriate scientific references. Mother cloning vectors, plant vectors and viral vectors are also commercially available. The cloning vector of Perbal (22) (pp, 277-29 8).

Many suitable microorganisms are American Type Culture Colle. (12301 Parklavn Drive, Rockville) , HD.

20852-1778).

From the disclosure of the present invention, one skilled in the art will appreciate that the present invention , or properties that are considered to be substantially functionally equivalent. It should be clear that a nucleotide sequence is intended. Similarly, books The invention provides promoter sequences having substantially the same function as those described herein. Also intended for columns. Thus, the present invention utilizes the functional elements described and disclosed herein. is intended to encompass, and in fact encompasses, functional isobars.

References 1, Grossman+ A, D, Er1ckson, J, W, and Gro ss, C, A,: r Escherichia coli h t p l? , the gene product is heat-shot is a sigma factor for the search promoter (ThehtpRGene Pro reduce of E, colt is a Sigma Factor for Heat-5hock Promoters) J, Ce1l, 38, 383-390, 2, 5traus, D, B,, vatter, V, A, and Gross, C.A.: The heat shock response in E. coli is σ32 (The Heat 5hock Re5po nseIn　E,co! 1 is Regulated by Changes In the Concentration or (732) J, Natu re, 329, 3411-351, 3, Ba old, Hl, Echols * H,,5traus,D,B,,Court v D,,Crowl,R , and Georgopoulos.

C, D: Through stabilization of σ32 by f phage lambda cm protein Induction of heat shock response in E. coli (Induction or the He at 5hock Ra5ponseof E, colITthrough 5 tabilization of a 32 by the Phage La mbdacll Protein) J, Genes & Dev,, l, 57-64, 19874, Ng, H., Ingrahas, J. L., and Marrv A, G: “Injury and inhibition in Escherichia coli as a result of growth at low temperatures. Dasage and Derepresslon in Esche rlchia colIResulting from Grovh at L ow Temperatures) J,” 9 Bacterol,, 84, 331-339, 19625. Hew, c, t,, 5cott, G, , and Davies, P. L.: Molecular biology of cryoprotection. “Raw production at low temperatures” Existence: Physiological and biochemical adaptations (Molecular Bjology or ^ntlrreeze, In Living In the Co1d: P hysiologicaland B1ochem! cat Adaptati ons) J, H, C, Heller ed., Efsvler 5science Publishing Co, Inc, N, Y, +pp.

117-123.1986 6, Duman, J., and Horvath *K: Cold tolerance of insects. The role of hemolymph proteins in 1esolymp h　ProteIns　in the　Co1d　Tolerance　ofI nsects) J, Ann, Rev, Physio! ,, 45.261- 270゜7, Nakamura, K., Masui, K3, and Inoyu e.

M.: [Transcriptional regulatory switch for constitutive 1pp gene expression in E. coli and Use of the lac promoter-operator fragment as romoter-OperatorFragsent as a Transc rlptlonal Control 5w1tch forthe Expr Ession of’ the Con5tltutjve Ipp Gene ! nEscherichia colt) J, J, Mo1. Appli ed Gen, ■, 289-299.1982 8, Vlasuk, G, P,, Inouye t S,, Ito, H,, I Takura, K. and Nouye, M.: Lipotan in Escherichia coli. Effect of complete removal of basic amino acid residues from signal peptide on protein secretion (EfTects or Complete Removal of’ Ba5i c As1no Ac1d Re5tdues from theSlgnal Pepticle on 5 creation of Lipoprotei n 1n Escherichia coll) J, J, Bjol, Che w,, 258, 7141-7148, 1983 9. Lehnhardt, S., POllItt, S., and Inouye, Hl: “Escherichia coli OmpA signal peptide effect on two hybrid proteins The Differential Effects of Deletion Mutations in the Hydrophobic Region of 1Jfect on Two Hybrid Proteins or Del etion Mutations vjthin the Hydrophobi c Reglon or the Escherlchia coil Osp ASignal Peptide) J, J, Blol, Chet, 262 , 1716-1719, 10, 1 nouye * S, , 5oberon, X ,, Francesehinl sT3, Itakura, K, and 1 nouye + M: "Signal peptide for membrane-separated protein secretion" Role of positive charges in the amino-terminal region of Charge at the Am1no Terlnal Reglon o rthe Signal Peptide for Protein 5ecr ation Across the Membrane) J, Proc, Nat l, Acad, Sei, USA, 79.343g-3441, 1982 11. Chuo, P. Y., and FaSIan, G. D.: r protein structure. Empirical Predictions or Prot eln Conformation) L Ann, Rev, Bioche*, 1 47. 251-27 [i, 1978 12, Messing, J., Crea * R, and Seeurg.

P, H,:r Shotgun DNA sequencing system (A 5ystes) for Shotgun DNA Sequencing) J, Nuclei c^cids Res, 9, 309-321. 19811B, Yanis ch-Perron, C., Vjeria%J, and Messlng,” , two improved M13 phage cloning vector and host strain: M13m Nucleotide sequences of p18 and pUc19 vectors (Improved M1 3Phage Cloning Vectors and Ho5t 5tra ins: Nucleotide Sequences or the M13+ +p18 and pUc19 Vectors) J.

Gene, 38, 103-119, 198514, Kreuger, J.H. and Walker, G.C.: groEL and dr+aK genes in E. coli. Induction by UV irradiation and nalidixic acid in offspring and htpR+ Genes of Escherich (groEL and dnaK) la colt and Induced by UV Irradiatlon and Na1id1xic Ac1d 1n ahtpR+-Depend entFashlon) J, Proc, Natl, Acad.

Sci, LISA, 81, 1499-1503, 198415, 0'Pa rrrell, P, H: High-speed two-dimensional electrophoresis of r proteins () 11gh Re 5olutjor+　Two-Dimensiona! Electrophor esis or Proteins) J, J, Blol, Chew.

250, 4007-4021. 197516, Feeney, R, E and and Burehas, T.S.: anti-freeze glycoprotein from boreal fish blood (^ ntlfreezeGlycoproteins from Po1ar Fi sh Blood) J, Ann, Rev.

Biophys, Chew, 15, 59-78, 198617, Inok uchl, K1, Furukava + H,, Nakamura sK, and Mizushiia, S.: Promoter region of r o m p f Characterization of in vitro deletion mutagenicity of positively regulated E. coli genes. Characterization by Deletion Mutag enesls in Vitro or the PromoterRegio n or o*pf, a Po5tively Regulated Ge ne of Escheriehia colt)], J, Mat, B4o ,, 178, 653-[K68, IL DeVrles+ A, L,: f cold Antifreeze peptides and glycopeptides in aquatic fish (^ntif’reeze Peptides and Glycopeptldes 1n Co1d-Wa ter　Fishes) J, Ann, Rev.

Physiol, 45, 245-60, 198319, Inouye, S. and 1 nouye, N, :r oligonucleotide using double-stranded plasmid DNA. Reotide-directed site-directed mutagenesis (011, gonueleotlde-D lrected 5ite-8specific Mutagenesis us ing Double-8tranced Plaslid DNA) J, 5 ynthesis and AppHeatphones or DNA and R NA 5ynthesis, edited by S, ^, Nara, ng (Acadesl cPress Inc, 0rlando), pL 181. -206, 19 8720, Inouye, S, and Inouye *M, :r of Escherichia coli Up promoter mutation in the lpp gene (Llpprosoter Mutations in the lppgeneofEscherjchi a colt) J, Nucleic Ac1ds Re5earch.

Vol, 13, N059, pp, 3103-3110, 198521, Ma Sui, Yl, Colellan, J, and Inouye *X,: “Gene Experimental Manipulation of Expression Gene Expression) J, Academy Press+In ouye, 1983 22, Perbal, B.: Practical Guide to Single Molecule Cloning (A. Prac tical Gu! de to Mo1ecular Clonlng) J, John Wllley and 5oz+ Wllley-1nterscie nce, New York *NY, 198g 23, ^subel, F. M. et al. 2 [Recent protocols in molecular biology ( (Current Protocol Mo1e) cular Biology (Current Protocols)) J, Broomlyn, NY and Village and 5onsIntersc ience, New York *NY (Supplements 1- 5), 24, Broeze, R.J., Solomon, C.J., and Pope. .

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pp, 383-389, 1982 International investigation failed international search report

Claims (39)

[Claims] 1. Synthesis is induced in E. coli only at temperatures below the normal growth temperature of E. coli. Get, protein. 2. A claim that shows stability at and below the normal growth temperature of E. coli. The protein award described in item 1. 3. The protein according to claim 1, which is a cytoplasmic protein. 4. 2. The protein of claim 1, having a molecular weight of about 7.4 kdal. 5. According to claim 1, it is possible to reduce the adverse effects of low temperature on viable E. coli cells. Proteins listed. 6. The protein according to claim 5, having the following amino acid sequence. [There is an array] 7. 7. A protein according to claim 6, which does not contain an initiating Met residue in its amino acid sequence. 8. 7. Protein according to claim 6, which is highly hydrophilic. 9. The protein according to claim 6, having a secondary structure indicative of an α-helical structure. Pak quality. 10. Gene transcription can be initiated at temperatures below the normal growth concentration of E. coli. promoter sequence. 11. Contains proteins that can reduce the negative effects of low temperatures on viable E. coli cells. The promoter according to claim 10, which is capable of initiating transcription of the encoded gene. -. 12. Contains proteins that can reduce the negative effects of low temperatures on viable E. coli cells. transcription of the coded gene can be initiated at a temperature below the normal growth temperature of E. coli. 12. The promoter according to claim 11, which is capable of. 13. Claim 12, wherein the transcription of the gene whose transcription is initiated at low temperature is the same gene. Promoter described in. 14. 14. The promoter according to claim 13, wherein the gene is cs7.4. 15. transcription of a heterologous gene instead of a homologous gene at the normal growth temperature of E. coli. 12. A promoter according to claim 11, which can be initiated with. 16. Upstream of the gene, located between nucleotides 1 and 605 shown in Figure 7. 13. The promoter according to claim 12. 17. DNA fragment shown in FIG. 18. DNA fragment from nucleotides 1 to 997 shown in FIG. 19. In the DNA fragment shown in Figure 7, the negative effect of low temperature on viable E. coli cells. A gene encoding a protein that can reduce the transcription of the gene at low temperature. Nucleotide containing promoters and other genetic regulatory elements that can be initiated by Punctuation sequence. 20. Encodes a protein that reduces the negative effects of low temperatures on growing E. coli cells DNA sequence. 21. 21. A DNA sequence according to claim 20, having the following nucleotide sequence: [There is an array] 22. can encode a protein whose transcription is cold induced in E. coli. A gene that is regulated by a promoter that is conductive. 23. The gene according to claim 22, which encodes the protein according to claim 1. 24. The gene according to claim 22, which encodes the protein according to claim 4. 25. The gene according to claim 22, which encodes the protein according to claim 5. 26. The gene according to claim 22, which encodes the protein according to claim 6. 27. 23. The gene according to claim 22, designated cs7.4. 28. Its transcription is also regulated by promoters other than cold-inducible promoters. The gene according to claim 22. 29. The promoter other than the low temperature inducible promoter is the lac promoter. , the gene according to claim 28. 30. 30. Encoding a protein at the normal growth temperature of E. coli. Genes listed. 31. A recombinant plasmid capable of expressing the protein according to claim 1. . 32. 32. The plasmid of claim 31, comprising the gene shown in FIG. 33. The requested protein also contains a promoter region sequence capable of initiating transcription of the gene. 33. The plasmid according to claim 32. 34. D upstream of the promoter region that can bind transcriptional regulatory proteins 34. The plasmid of claim 33, further comprising an NA sequence. 35. 35. The plus of claim 34 further comprising a regulatory protein binding site. Mid. 36. capable of expressing the protein of claim 1 from an extrachromosomal element; Enterobacteriaceae cells. 37. 37. The cell of claim 36, wherein the cell is E. coli. 38. It can only be induced and proliferate at temperatures below the normal growth temperature of E. coli. A protein production method that can reduce the negative effects of low temperatures on E. coli bacteria and a promoter capable of initiating transcription of the gene encoding the protein. A method for manufacturing E. coli at an exponential rate to a desired growth level. growing at a target temperature, lowering the temperature to approximately 15°C or less, and It consists of allowing the bacteria to continue growing at that temperature, thereby increasing the protein content. synthesis, suspending growth of the culture, and encoding the protein. It contains at least a gene to be encoded and a promoter sequence that initiates transcription of the gene. isolating therefrom a desired DNA fragment. 39. Claim 3, wherein the gene is cs7.4 and the protein is csp7.4. 8. The manufacturing method described in 8.