CN103435689B - Molecular chaperone TSPcpn47 and polynucleotide encoding same - Google Patents

Abstract

The invention discloses a molecular chaperone TSPcpn47 and a polynucleotide encoding the molecular chaperone. The molecular chaperone TSPcpn47 is derived from the bacteriophage TSP4 of Thermus thermus, which can stabilize the protein structure and prevent protein aggregation and denaturation, and improve protein production. stability, and its nucleotide sequence can be used to construct genetically engineered strains producing this molecular chaperone.

Description

A molecular chaperone TSPcpn47 and polynucleotide encoding the molecular chaperone

technical field

The present invention relates to a molecular chaperone TSPcpn47 and a polynucleotide encoding the molecular chaperone. The molecular chaperone stabilizes nascent proteins in organisms, assists other proteins in correct folding, and improves protein stability, and belongs to the field of biotechnology. the

Background technique

Molecular chaperones are a family of proteins that are very conserved in evolution. They can non-specifically bind proteins of different structures, sizes and functions and catalyze the formation of specific conformations of these proteins. They play a role in stabilizing new proteins and assisting other proteins in organisms. Correct folding, assembly, transport and even degradation. Under stress conditions, molecular chaperones can stabilize protein structure and prevent protein aggregation and denaturation. They also have the function of repairing denatured proteins, which is of great significance to the function of proteins. the

The first molecular chaperone - nucleoplasmin (nucleoplasmin) was discovered by Laskey et al. in the leaching solution of Xenopus laevis eggs in 1978. They must study the process of histone binding to DNA to form nucleosomes. Their assembly depends on the participation of an acidic nucleoplasmin. In vitro experiments also showed that only by mixing histones with excess acidic nucleoproteins can they be assembled with the added DNA to finally form a nucleosome structure, but there is no acidic nucleosomes in the formed nucleosomes. When only DNA and histones are present, they cannot self-assemble into nucleosome structures, but form precipitates. the

Most of the molecular chaperones discovered so far belong to the category of heat shock protein (HSP), which can be roughly divided into chaperonin (Cpn) family , heat shock protein 70 family (HSP 70 family), heat shock protein 90 family (HSP 90 family) and heat shock protein 100 family, and other conserved protein families, have not yet been derived from Thermus Molecular chaperones of bacteriophages have been reported. Molecular chaperones have industrial application value because they can improve the stability of proteins (enzymes). the

Contents of the invention

The present invention aims to provide a molecular chaperone TSPcpn47, the molecular chaperone TSPcpn47 is derived from the bacteriophage TSP4 of Thermus thermophila, and its amino acid sequence is shown in SEQ ID NO:1. the

Another object of the present invention is to provide a polynucleotide encoding molecular chaperone TSPcpn47, the nucleotide sequence of which is shown in SEQ ID NO:2. the

Beneficial effects of the present invention:

The molecular chaperone TSPcpn47 of the Thermus phage TSP4 obtained in the present invention can independently perform the function of a molecular chaperone, that is, to help denatured protein refolding, and prevent other proteins from denaturing under high temperature, acidic conditions, etc., and can be used in high temperature and other environments It assists the functional protein to maintain its activity, reduces protein denaturation, and plays an important role in protecting the physiological activity of the functional protein. Its nucleotide sequence can be used to construct genetically engineered strains that can catalyze the formation of the above products.

Description of drawings

Fig. 1 is the cloning fragment electrophoresis schematic diagram of molecular chaperone TSPcpn47 gene of the present invention;

Fig. 2 is a schematic diagram of analysis of the conserved domain of the molecular chaperone TSPcpn47 gene of the present invention;

Figure 3 is a schematic diagram of enzyme digestion of molecular chaperone TSPcpn47 gene and pET-28a(+) of the present invention, wherein swimming lane 1 is the product recovered from enzyme digestion gel of molecular chaperone TSPcpn47 gene, swimming lane 2 is pET-28a(+), and swimming lane 3 is Marker ;

Fig. 4 is the SDS-PAGE analysis detection electrophoresis figure of molecular chaperone TSPcpn47 gene expression protein of the present invention, and its size is 29000 Daltons, wherein swimming lane 1 is Protein Marker; Swimming lane 2 is the supernatant after Ro-pET28a is broken; -pET28a- TSPcpn47 crushed supernatant; lane 4 is the precipitate of Ro-pET28a crushed; lane 5 is the precipitate of Ro-pET28a- TSPcpn47 crushed.

Figure 5 is the SDS-PAGE analysis and detection electrophoresis of molecular chaperone TSPcpn47 of the present invention, wherein lane 1 is Protein Marker; lane 2 is the supernatant after fragmentation of pET28a- TSPcpn47 /Rosetta; lane 3 is the supernatant after fragmentation of pET28a- TSPcpn47 /Rosetta Clear His-trap column; Swimming lane 4 is 10mM imidazole eluent; Swimming lane 5 is 150mM imidazole eluent; Swimming lane 6 is 500mM imidazole eluent; Swimming lane 7 is 500mM imidazole eluent;

Fig. 6 is a schematic diagram of the protein content standard curve of the present invention;

Fig. 7 is a schematic diagram of the phosphate concentration standard curve of the present invention;

Fig. 8 is the influence of the temperature of the present invention on the activity of molecular chaperone TSPcpn47;

Fig. 9 is the assisting effect of molecular chaperone TSPcpn47 of the present invention on the recovery of GFP fluorescence intensity;

Fig. 10 is the effect of molecular chaperone TSPcpn47 of the present invention on the thermal stability of GFP.

Detailed ways

The present invention will be described in further detail below by the examples, but the content of the present invention is not limited thereto, the methods in the present embodiment are all operated according to conventional methods if there is no special instructions, and the reagents used are conventional reagents if there are no special instructions or according to Reagents configured by conventional methods. the

Embodiment 1: the amplification of molecular chaperone TSPcpn47 gene (using the Thermus phage TSP4 genome DNA as model

plate)

(1) The primer sequences used for the amplification of the TSP4 phage molecular chaperone TSPcpn47 gene are as follows:

Forward primer: 5'- CG GAATTC ATGGCACTCTTATCTAAC -3'

Reverse primer: 5'- ACAT CTCGAG CCATGAGGCCACCTCCT -3'

(2) The amplification system is as follows:

Table 1: Amplification reaction system components

                                                    

(3) The amplification conditions are as follows:

Mix the reaction system evenly, pre-denature at 95°C for 4 minutes, then denature at 95°C for 30 s, anneal at 60°C for 45 s, and extend at 72°C for 90 s. After 30 cycles, extend at 72°C for 10 min. After the reaction, take 2 μl of the product , Electrophoresis analysis was carried out in 10g/L agarose gel (see Figure 1).

Example 2: Gel recovery and purification of PCR products

①Create 1.0% agarose gel in the electrophoresis apparatus;

② Apply electrophoresis to the PCR product to be separated and purified, and stop the electrophoresis at an appropriate position;

③Cut off the gel containing the target fragment under ultraviolet light, and transfer it to a 1.5ml Ep tube;

④Recover the target fragment with the Gel Recovery Kit of Soleil Biotechnology Co., Ltd., and the recovery method is operated according to the instructions.

The gene sequence was submitted to the NCBI database Conserved Domain Database protein conservative sequence analysis software for analysis, and the dCTP deaminase analysis results are shown in Figure 2. the

Embodiment 3: Construction of recombinant expression vector

(1) Preparation of the linear vector pET-28a(+) with cohesive ends and the target fragment

In order to connect the target gene fragment to the expression vector pET-28a(+), it is necessary to make the target fragment have a fragment with sticky ends, that is, a restriction site. Similarly, in order to enable the target fragment to be inserted into the vector, it is also necessary Make vectors with cohesive ends and make their restriction sites the same.

A. Extraction of pET-28a(+) plasmid: use the plasmid extraction kit (Soleibao), the operation steps are as follows:

Strain activation: Dip the Escherichia coli strain containing the pET-28a(+) plasmid frozen at -80°C with a sterile inoculation loop, inoculate it on the ampicillin LB plate with the third-line method, and incubate at 37°C for 12-16 hours;

② Enrichment and collection of bacteria: Take 5 μl of ampicillin (final concentration 100 μg/ml) and add it to 5 ml of LB medium; pick positive clones with an inoculation loop and inoculate them in Kan ⁺ -LB medium; then shake at 37°C Cultivate overnight on a bed at 150 rpm, centrifuge the cultured bacterial solution at room temperature at 12,000 rpm for 1 min to precipitate the bacterial cells, and discard the supernatant;

③ Add the solution to the centrifuge tube (contains RNase) 250 μl, shake until the bacteria are completely suspended, and transfer to a 1.5ml EP tube;

EP tube adding solution 250 μl, gently invert several times, and place at room temperature for 3 minutes to degrade the RNA in solution I by RNase, then add solution 350 μl, centrifuge at 13000 rpm for 10 min at room temperature;

The supernatant obtained after centrifugation was transferred to a DNA adsorption column, centrifuged at room temperature, 12000 rpm, 1 min, recovered twice, and the waste liquid was discarded;

Add 500 μl of washing solution to the adsorption column, and centrifuge at 12,000 rpm for 1 min at room temperature;

⑦ Then add 750 μl rinse solution, centrifuge at room temperature for 1 min at 12,000 rpm, discard the waste solution (repeat once); centrifuge again at room temperature, at 13,000 rpm, for 2 min;

⑧ Move the adsorption column into a new 1.5 ml centrifuge tube, add 70 μl of eluent preheated at 65°C to the center of the adsorption membrane, let it stand at room temperature for 2 minutes, then centrifuge at 12000 rpm for 1 minute at room temperature, discard the adsorption column, and obtain the plasmid ;

⑨ Take 1 μl of the obtained plasmid for electrophoresis detection and quantification.

B. Enzyme digestion identification of plasmid pET-28a(+) and target fragment

Table 2: Reaction System Components

    

Incubate at 37°C for 6 hours, detect the double-digestion products by 1% agarose gel electrophoresis, and recover the double-digestion products with Solebol Gel Recovery Kit (see Figure 3).

(2) Connect the molecular chaperone TSPcpn47 gene fragment of TSP4 phage to the pET-28a(+) vector to obtain a recombinant plasmid

①The connection reaction system is as follows:

Table 3: Reaction System Components

②Conditions: 16°C, connect overnight.

(3) Transformation of recombinant plasmids

In 100 μl of competent cell DH5a, add 10 μl of the ligation product, ice-bath for 30 minutes, then heat shock at 42°C for 2 minutes, and then ice-bath for 2 minutes, then add 400 μl of LB liquid medium, shake and culture at 37°C for 1 hour at 150 rpm, and then apply An LB solid plate containing ampicillin (kan ⁺ ) was placed, and cultured upside down at 37°C for 16 hours.

(4) Detect strains containing recombinant plasmids by colony PCR, and screen out positive colonies

① Observe the transformation plate, pick several colonies at random, pick up with a 10μl pipette tip, dissolve in 20μl ddH ₂ O, mix well, transfer 10μl of the bacterial solution to the LB liquid medium, and use the remaining 10μl of the bacterial solution for thermal lysis. Cracking temperature 98°C, time 10min;

② After lysing, use a handheld centrifuge to shake gently, and the lysate is used as a template for colony PCR;

③ Colony PCR amplification reaction system is as follows:

Table 4: Reaction System Components

④ Colony PCR reaction conditions

After mixing the reaction system, pre-denature at 95°C for 4 minutes; then denature at 95°C for 45 s, anneal at 60°C for 45 s, and extend at 72°C for 90 s, 30 cycles; extend at 72°C for 10 min; take 2 μl of the product after the reaction is completed , carried out electrophoresis analysis in 1% agarose gel;

⑤ Positive colonies were identified and sent to Kunming Shuoyang Biotechnology Company for sequencing.

(5) Positive clone enrichment

① Add 5 μl of kanamycin (final concentration 100 μg/ml) into 5 ml of LB medium;

②Pick positive clones with an inoculation loop and inoculate them in Kan ⁺ -LB medium;

③ Then put it into a shaker at 37°C at 180rmp and incubate for 6-8h.

Recombinant plasmid extraction (using the method provided in the plasmid extraction kit)

①Collect bacteria: Add the bacteria cultured in step (5) into a 5ml centrifuge tube, collect the bacteria precipitate by centrifugation, and discard the supernatant;

② Add 250 μl of solution I (containing RNase), shake until the cells are thoroughly dispersed, and transfer to a 1.5ml EP tube;

③ Add 250 μl of solution II and mix gently to make the RNase in solution I digest the RNA in the bacteria;

Add 350 μl of solution III, mix gently, and centrifuge at 12000 rpm for 10 min;

⑤ Transfer the supernatant to a DNA adsorption column, place at room temperature for 2 min, mix well, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid in the collection tube;

⑥ Add 700 μl rinse solution to the adsorption column, centrifuge at 12000rpm for 1min, discard the waste solution (repeat once);

⑦ room temperature 13000rpm, centrifuge again for 2 minutes, and leave the adsorption column open for several minutes to remove the remaining rinse solution;

⑧ Move the adsorption column into a new 1.5ml centrifuge tube, add 70 μl of eluent preheated at 65°C to the center of the adsorption membrane, place it at room temperature for 1 min, then centrifuge at 12000 rpm for 2 min at room temperature, and repeat it again to increase the recovery rate;

⑨ Take 1 μl of the obtained plasmid for electrophoresis analysis.

(6) Enzyme digestion identification of recombinant plasmids

①The enzyme digestion system is as follows:

Table 5: Reaction System Components

②Enzyme digestion conditions: 37°C, 3h, take 2μl and run agarose gel electrophoresis.

(7) The recombinant plasmid pET28a- TSPcpn47 was transformed into the expression strain Rosetta (DE3)

In 100 μl of competent cells Rosetta (DE3), add 3 μl of recombinant plasmid, ice-bath for 30 minutes, then heat shock at 42°C for 1-2 minutes, then ice-bath for 2 minutes, then add 400 μl of LB liquid medium, culture at 37°C, 150rpm shaker After 1 hour, LB solid plates containing ampicillin (Kan ⁺ ) were coated, and cultured upside down at 37° C. for 12 hours.

(8) Induced expression and purification of molecular chaperone TSPcpn47

① Induced expression of TSP4 phage chaperone TSPcpn47 in Rosetta (DE3)

The constructed recombinant vector pET28a- TSPcpn47 was transformed into the expression host strain Rosetta (DE3), the strain containing the recombinant plasmid was cultured overnight at 37°C, and the bacterial solution was inoculated into 100ml Kan+-LB (final concentration of ampicillin 100μg/ ml) in the culture solution, cultured on a shaker at 37°C until its OD ₆₀₀ was 0.6; 1ml of the bacterial solution was taken out as a control experiment; the rest of the bacterial solution was added to 100mM IPTG stock solution to a final concentration of 1mM, and cultured on a shaker at 90 rpm to induce the expression of 8 Hour.

SDS-PAGE detection of TSP4cpn47 protein

Take out a small amount of induced bacterial liquid to measure the OD value, take different amount of bacterial liquid according to the different OD value of the bacterial liquid to make the amount of bacterial cells equal, centrifuge at 12000rpm for 5min, discard the supernatant; add 80 μl ddH ₂ O, 20 μl 5× Loading buffer solution, shaking to disperse the bacteria; thermally lyse at 98°C for 10 minutes to release the protein from the bacteria; load the sample on the electrophoresis instrument at 100V, 60mA, electrophoresis for 2 hours, add 100ml R250 Coomassie brilliant blue Stain with the staining solution, shake on a shaker, 100 rpm, after 20 minutes; pour out the staining solution, rinse with water, then add an appropriate amount of destaining solution at 100 rpm, for 10 hours, and finally scan the gel with a scanner to detect protein expression (see Figure 4 ), the result was a target protein band with a size of about 29kD, and TSPcpn47 was expressed in both the supernatant and the precipitate.

②Purification of recombinant helicase protein

Use the above method to induce a large number of Rosetta strains containing the recombinant plasmid pET28a- TSPcpn47 , collect the bacteria by centrifugation (4°C, 5000g, 10 min), and suspend the bacteria in an appropriate amount of buffer (to make the OD ₆₀₀ of the bacteria suspension reach 20) A [20 mM sodium phosphate, 500 mM NaCl, 30 mM imidazole, pH 7.8], sonicate the cells on ice, centrifuge at 10,000 g for 15 min at 4°C, and load the supernatant on the His Trap equilibrated with buffer A In the HP column (1 ml, GE Healthcare), the bound protein was eluted with 20 ml of buffer B [20 mM sodium phosphate, 500 mM NaCl, 30 mM imidazole, pH 7.4] containing imidazole gradient (0-500 mM), The eluate was collected in sections, detected by SDS-PAGE, and finally dialyzed overnight with buffer [50 mM Tris-HCl, 2 mM Mg2+, pH7.4] to obtain recombinant protein TSPcpn47, and the purified recombinant protein fraction was subjected to SDS- PAGE analysis (see Figure 5).

(9) Explore the soluble expression conditions of the recombinant molecular chaperone TSPcpn47

The expression strain pET28a- TSPcpn47 /Rosetta was cultured at 28°C, and the expression of the target protein was induced by 1mM IPTG. The pET28a/Rosetta transformed into the pET-28a plasmid without the target fragment was used as a control, and the bacteria were collected by centrifugation, and the cells were sonicated. After centrifuging again, the supernatant and the precipitate were collected respectively, and the expression of the recombinant protein in the supernatant and the precipitate was detected by SDS-PAGE (see Figure 5).

(10) Determination of recombinant protein TSPcpn47 content

Protein content was determined by Bradford method, reagents: Coomassie Brilliant Blue G-250 stock solution, 1 mg/mL BSA standard protein solution; buffer: pH 8.0, 50 mmol/L Tris-HCl buffer.

The steps are: take seven 1.5ml effendorf tubes and add BSA, sample solution to be tested, deionized water, and Coomassie Brilliant Blue G-250 stock solution respectively, mix well within 5-20 min, and measure the wavelength with a UV spectrophotometer. For the absorbance value at 595 nm, the BSA solution with a concentration of 0 mg/mL was used as a blank control, and the specific method is shown in the table below. the

Table 6: Components and dosage

Draw a standard curve according to the determination results of standard proteins with different concentrations. Take the protein concentration as the abscissa and the OD value as the ordinate to draw the standard curve (see Figure 6). According to the OD value of the unknown protein, the protein can be detected on the standard curve. concentration.

Calculation formula:

Protein concentration of the sample to be tested (mg/ml) = OD value of the test sample/OD value of the standard protein × concentration of the standard protein × dilution factor

Use this method to determine the content of recombinant protein TSPcpn47 in the protein purification solution: the measured OD595 is 0.093, and the protein concentration is 0.0697 μg/μL when substituted into the protein standard curve equation.

(11) Detection of ATPase activity of molecular chaperone TSPcpn47

The ATPase activity of the molecular chaperone TSPcpn47 was detected using the ATPase detection kit from BioAssay Systems.

① Preparation of phosphate concentration standard curve

Prepare 800μl phosphate premix solution with a phosphate concentration of 50μM (mix 40μl 1mM phosphate standard solution with 760μl ddH2O), prepare a clean 96-well plate, add samples according to the following table, and obtain the phosphate concentration standard through the detection of the kit The curve is shown in Figure 7.

② Detection of ATPase activity

Set the reaction volume to 40uL, put the enzyme sample, inactivated enzyme and Tris-HCl buffer control into different wells respectively

Prepare three parallel samples and place them at 37°C, 45°C, and 55°C for 30 minutes, then add 200 μL of chromogen to terminate the reaction, let stand at room temperature for 30 minutes, and read the OD value at a wavelength of 630 nm.

Definition of enzyme activity: One unit of enzyme activity is equivalent to the amount of 1 μmol phosphate ion catalyzed per milligram of protein per minute under the detection conditions, defined as 1U. According to the definition of enzyme activity, its ATPase enzyme activity is 1.36U. the

③The effect of temperature on molecular chaperone TSPcpn47

Similarly, using the reagents and methods provided by the ATPase activity assay kit, with a gradient of 10°C, the effects of different temperatures on the ATPase activity of the recombinant protein chaperone TScpn47 were measured (see Figure 8). °C has ATPase activity, and 60 °C is the optimum temperature for enzyme activity.

(12) Molecular chaperone TSPcpn47 helps GFP restore fluorescence experiments (see Figure 9)

① Take U2OS cells in good condition and add them to a 6-well cell culture plate, and replace with DMEM high-glucose medium (original Gibco, USA) without double antibody and serum 4 hours before transfection;

② Preparation of transfection solution

Dissolve 5 μg of pEGFP plasmid in 200 μl DMEM;

Dissolve 10 μl of Lipofect transfection reagent in 200 μl DMEM;

③The pEGFP plasmid solution was mixed with the lipofect solution respectively, and then allowed to stand at room temperature for 20 minutes, then evenly added to the cell culture medium, and cultured at 37°C, 5% CO ₂ for 12 hours;

④After 24 hours, discard the old culture medium, wash the cell surface twice with 1×PBS, add an appropriate amount of 0.25% trypsin, shake the cell culture flask to make the trypsin evenly cover the cells, and let it stand for digestion at 37°C until the monolayer of cells changes. If it becomes blurred and small cracks appear, gently pat until all the cells fall off;

⑤Collect 1mL of cell suspension (trypsin treatment), centrifuge at 4°C, 1000rpm for 2min, discard the supernatant, resuspend the pellet with 1mL of 100 mM Tris-HC1 (pH7.8) buffer, and then ultrasonicate for 3min ( 15% power, crushing for 5s, pause for 3s), after the crushing is over, put it on ice for standby;

⑥ Add HCl to the green fluorescent protein GFP (10mM) to a final concentration of 0.1mM, and denature at room temperature (about 25°C) for 1 h;

⑦ Dilute 100-fold refolding with buffer (100 mM Tris-HC1, 40 mmol/L MgC12, pH7.8), and add molecular chaperone TSPcpn47 (final concentration 0.2 μg/μL) and ATP (final concentration 4.0 mM) to add The renaturation situation of GFP after bovine serum albumin (BSA) of equal substance concentration, and the dilution renaturation situation of GFP itself are used as controls;

⑧Using a fluorescent microplate reader Fluostar Optima, excitation at 480nm, emission at 520nm, real-time measurement of fluorescence changes, with 0.1 mol/L Native GFP fluorescence intensity as 100%, the results are shown in Figure 9, TSPcpn47 can significantly increase the green fluorescent protein after acid denaturation Refolding effect, has the ability to assist in restoring its function. the

(13) Effect of molecular chaperone TSPcpn47 on the thermal stability of GFP

①Mix the molecular chaperone TSPcpn47 and GFP protein at a molar ratio of 1:1, and then add ATP to a final concentration of 4mmol/L;

②Incubate at 60°C for 60 min, take a sample every 10 min, measure the residual fluorescence intensity, and take the enzyme activity at 0 min as 100%;

③ Take the thermal stability of GFP at 60°C after adding bovine serum albumin (BSA) in an equimolar ratio, and the thermal stability change of GFP protein at 60°C, that is, the change of fluorescence intensity as a control. The results are shown in Figure 10, TSPcpn47 It can significantly improve the thermal stability of green fluorescent protein, and assist the functional protein to maintain its physiological activity in a high temperature environment.

sequence listing

<110> Kunming University of Science and Technology

<120> A molecular chaperone TSPcpn47 and a polynucleotide encoding the molecular chaperone

<160> 4

<170> PatentIn version 3.5

the

<210> 1

<211> 204

<212> PRT

<213> Phage TSP4

the

<400> 1                                                          

1 5 10 15

Val Ile Leu Gln Asp Gly Gln Thr Ile Leu Lys Pro Ser Pro Phe

16 20 25 30

Leu Gln Ala Leu Leu Thr Ala Lys Glu Gly Lys Arg Val Gly Leu

31 35 40 45

Val Leu Asp Glu Ile Asn Arg Gly His Pro Thr Ala Leu Asn Arg

46 50 55 60

Val Phe Arg Leu Leu Ala Gln Arg Gln Phe His Ser Asp Trp Tyr

61 65 70 75

Gly Leu Leu Asp Leu Lys Gly Lys Asp Leu Val Val Ile Ser Thr

76 80 85 90

Ala Asn Ala Gly Val Glu Tyr Thr Val Ser Lys Leu Asp Arg Ala

91 95 100 105

Met Arg Glu Arg His Leu Trp Ile Tyr Val Pro Arg Pro Asp Pro

106 110 115 120

Ser Val Val Phe Thr Val Leu Gln Glu Arg Val Pro Asn Leu Thr

121 125 130 135

Pro Ala Gln Ala Glu Val Ile Thr Asn Leu Tyr Ala Ala Ser Asp

136 140 145 150

Gln Leu Leu Gly Val Arg Glu Ser Leu Asp Ile Ala Tyr Leu Leu

151 155 160 165

Ala Asn Gly Val Asp Leu Gln Ser Ala Val Glu Leu Thr Val Lys

166 170 175 180

Gly Ala Ala Tyr Val Ser Asn Thr Ala Leu Glu Val Ala Asp Gly

181 185 190 195

Leu Ile Ala Ser Ala Lys Ala Leu Thr

196 200 204

the

<210> 2

<211> 612

<212> DNA

<213> TSP4 phage

the

<400> 2

atggacattg gtggtctcat ggacccgcta gctattgaag gtacggtcat 50

cttgcaggac gggcagacca tccttaagcc ctctcccttc ctgcaagctc 100

ttttgacggc caaggaggga aaacgggtgg gtttggtgct ggatgagatt 150

aatcgcgggc atcccaccgc cctcaaccgt gtgttcaggc ttctggccca 200

gaggcagttc cactccgact ggtacgggtt gcttgacctc aagggcaagg 250

acctggtggt catttccacg gccaatgcgg gcgtagagta tacggttagt 300

aagcttgacc gggccatgcg tgagcggcac ctgtggatat atgttcccag 350

gcctgacccg tctgtagtgt ttaccgttct gcaggaacgg gtgccgaatt 400

tgacgcctgc acaagcagag gttattacca atctttacgc agcttcggat 450

cagcttctag gcgtgcgcga atctttggat atcgcctact tgctggctaa 500

tggtgtggac ctgcagtctg ctgtggaact cacggtcaag ggggcggctt 550

atgtcagcaa cacagccttg gaggtggcag acgggcttat tgctagcgct 600

aaggctctga cc 612

the

<210> 3

<211> 26

<212> DNA

<213> Artificial sequence

the

<400> 3

cggaattcat ggcactctta tctaac 26

the

<210> 4

<211> 27

<212> DNA

<213> Artificial sequence

the

<400> 4

acatctcgag ccatgaggcc acctcct 27

Claims (2)

1. a molecular chaperones TSPcpn47, is characterized in that: its aminoacid sequence is as shown in SEQ ID NO:1.

2. the polynucleotide of molecular chaperones TSPcpn47 described in coding claim 1, is characterized in that: its nucleotide sequence is as shown in SEQ ID NO:2.