CN110437331A - Method for improving inclusion body protein renaturation of smooth turtle shell serine protease inhibitor - Google Patents

Abstract

The present invention aims to provide a method for the renaturation and activity of the fusion protein TrxA -ApSerpin- FA72 inclusion body protein of soft-shelled turtle methyl-serine protease inhibitor. The present invention adopts the method of adding urea in the denaturing liquid, and using guanidine hydrochloride to dilute and renature to activate the fusion protein TrxA -ApSerpin- FA72 inclusion body protein of the smooth turtle methyl-serine protease inhibitor, and control the protein concentration in the whole protein renaturation process. 1-10μg/mL, the obtained recombinant protein TrxA- ApSerpin -FA72 has an inhibitory activity of more than 95% on serine protease at pH=7~9 and 60°C, and can effectively obtain an active recombinant protein TrxA -ApSerpin- FA72, which is Subsequent further research on the function of the smooth turtle methserine protease inhibitor lays the foundation.

Description

A method for improving the renaturation of the inclusion body protein of the smooth soft-shelled turtle methylserine protease inhibitor

Field of Invention

The invention belongs to the technical field of preparation of active proteins, and in particular, the invention relates to the technical field of a method for significantly improving the renaturation and activity of the inclusion body protein of the methyserine protease inhibitor of smooth turtle.

Background technique

Serine protease inhibitors are generally single-chain proteins composed of about 400 amino acid residues. The center of the protein is surrounded by 9 α helices and constitutes 3 β sheets. At the same time, its active reaction center loop (RCL) is generally far away from serine proteases. The center of the inhibitor is about 30-40 residues from the carboxyl terminus of the inhibitor. ApSerpin-FA72 is a member of the serine protease inhibitor superfamily, a Kazal-type protease inhibitor, which is involved in the negative regulation of serine protease hydrolysis cascades. Serine protease inhibitors also play a negative regulatory role in the activation of prophenoloxidase and the expression of antimicrobial peptides. They also inhibit the digestive enzymes of insects and regulate inflammation and blood coagulation.

Xinjiang is located in the hinterland of the Eurasian continent with arid climate. Among them, Coleoptera insects are the most representative class of insects. Anatolicapolitaborealis belongs to the Tenebrionodae family Tenyriini. Anatolica is a widely distributed desert insect, and the smooth soft-shelled turtle may have a unique immune regulation defense mechanism. Among them, serine protease inhibitors may play a key role in its unique immune regulation defense mechanism.

In the process of genetic engineering development, E. coli is used as a prokaryotic expression system. When exogenous genes are highly expressed in E. coli, high-density, insoluble protein particles will be formed, and at the same time, they will not have biological activity. biological activity. Therefore, when the protein is expressed in the form of inclusion bodies, the renaturation of the inclusion bodies is the key technology for protein production. Different inclusion bodies are treated with different detergents and denaturing agents, and they are washed, dissolved and renatured to obtain biological activity. protein.

At present, the existing patent CN109251936A has disclosed a preparation method of the fusion protein TrxA-ApSerpin-FA72 of the smooth turtle methaserine protease inhibitor. However, the constructed engineering strain pET32a-ApSerpin-FA72/BL21 was induced to express normally in Escherichia coli. Due to the high protein expression, the fusion protein mainly existed in the form of inclusion bodies. It is much smaller than the inclusion body protein. Therefore, in order to obtain a large amount of active high-purity serine protease inhibitor, the present invention provides a method for improving the renaturation and activity of the fusion protein TrxA-ApSerpin-FA72 inclusion body protein. Methods. It lays the foundation for the further study of the function of the smooth turtle methserine protease inhibitor.

SUMMARY OF THE INVENTION

Aiming at the technical problems of low purity and weak activity of the T. glabrata meth serine protease inhibitor prepared in the prior art, the present invention aims to provide a fusion protein TrxA-ApSerpin-FA72 inclusion body protein that significantly improves the complexation of the T. glabrata methaserine protease inhibitor fusion protein. methods of sexuality and activity. The invention provides a method for improving the renaturation and activity of the inclusion body protein of the fusion protein TrxA-ApSerpin-FA72 of the smooth turtle methaserine protease inhibitor, which can effectively enrich the high-concentration inclusion body fusion protein induced and expressed, and the protein activity is significantly improved after the treatment. It solves the technical difficulties reported by the current recombinant protein TrxA-ApSerpin-FA72, which is mainly an inactive inclusion body protein, and there is no related renaturation method that significantly improves the activity of the recombinant protein TrxA-ApSerpin-FA72 inclusion body protein. Further research on the function of the smooth turtle methase inhibitor will lay the foundation, and the study of its molecular mechanism will provide the basis for the unique immune regulation and defense mechanism of desert insects.

The present invention provides a method for significantly improving the renaturation and activity of the fusion protein TrxA-ApSerpin-FA72 inclusion body protein of the soft-shelled turtle methyl-serine protease inhibitor. The specific method steps are as follows:

(1) Transform the recombinant plasmid pET32a-ApSerpin-FA72 into Escherichia coli strain BL21 (DE3), screen positive single clones and cultivate them overnight at 37°C at 220 r/min, and inoculate them in LB medium at 1:100 the next day Medium (Amp ⁺ ) was cultured for 4h, the OD value of the bacteria was 0.6, added with different final concentrations of IPTG, induced at 32°C, 180r/min for 4h, centrifuged at 8000 × g for 10min to collect the precipitate, and resuspended the bacteria with phosphate buffer;

(2) Wash the precipitate with the prepared Tris-HCl buffer, fully dissolve the precipitate in the solution by ultrasonication for 2 min, centrifuge at 8000 × g for 10 min, repeat three times and wash the washing liquid remaining on the cells with Tris-HCl buffer net;

(3) The inclusion bodies were fully dissolved in the denaturing solution at 4°C for 1 h, centrifuged at 8000 × g for 15 min, and the renaturing buffer was slowly added to the lysing solution with a peristaltic pump within 18 h, and slowly stirred with a magnetic stirrer.

Preferably, the IPTG concentration is 0.4 mmol/L.

Preferably, the phosphate buffer is prepared by mixing 137 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L Na ₂ HPO ₄ and 2 mmol/L KH ₂ PO ₄ .

Preferably, the prepared Tris-HCl buffer is prepared by mixing 2mol/L urea, 50mmol/L Tris-HCl, 100mmol/LNaCl, 0.1mmol/L DTT, and 0.1mmol/L EDTA.

Preferably, the denaturing solution is prepared by mixing 50mmol/L Tris-HCl, 100mmol/L NaCl, 0.1mmol/L DTT, 0.1mmol/L EDTA, 6mol/L guanidine hydrochloride, 0.4mol/L L-Arg, pH=8.0 .

Preferably, the renaturation buffer is 50 mmol/L Tris-HCl, 100 mmol/L NaCl, 0.1 mmol/L DTT, and 0.1 mmol/L EDTA, prepared by mixing.

The strain pET32a-ApSerpin-FA72/BL21 used in the present invention has been disclosed in the prior art, and the existing patent CN109251936A has also disclosed a preparation method of the fusion protein TrxA-ApSerpin-FA72 of the smooth turtle methylserine protease inhibitor , the constructed engineering strain pET32a-ApSerpin-FA72/BL21 has become a well-known strain.

By implementing the technical scheme of the present invention, the following beneficial effects can be achieved:

The invention provides a method for significantly improving the renaturation and activity of the fusion protein TrxA-ApSerpin-FA72 inclusion body protein of the smooth turtle methaserine protease inhibitor. By adding urea to the denaturing solution and using guanidine hydrochloride to dilute and renature the fusion protein TrxA-ApSerpin-FA72 inclusion body protein, the protein concentration of the protein was controlled at 1 during the whole protein renaturation process. -10μg/mL, the obtained recombinant protein TrxA-ApSerpin-FA72 has an inhibitory activity of more than 95% on serine protease at pH=7～9 and 60℃, which can effectively obtain the active recombinant protein TrxA-ApSerpin- The FA72 protein solves the technical difficulties that the current acquisition of the recombinant protein TrxA-ApSerpin-FA72 is mainly an inactive inclusion body protein, and there is no relevant report on the renaturation method that significantly improves the activity of the inclusion body protein of the recombinant protein TrxA-ApSerpin-FA72, It lays the foundation for the further study of the function of the smooth turtle methserine protease inhibitor.

Description of drawings

Figure 1 shows the expression identification of recombinant protein TrxA-ApSerpin-FA72 and its expression analysis.

In the figure, A is the recombinant protein expression identification map, where M is the standard molecular weight protein, 1 is before pET-32a-ApSerpin-FA72/BL21 induction, 2 is after pET-32a-ApSerpin-FA72/BL21 induction; B is 32℃ , 0.6mmol/L IPTG induced protein expression form analysis recombinant protein expression form identification chart, in which 1 is the recombinant protein in the centrifuged supernatant after ultrasonication, 2 is the recombinant protein in the centrifugal precipitation after ultrasonication; C is TrxA-ApSerpin - Western blot identification chart of FA72, where M is the standard molecular weight protein, and 1 is the recombinant protein TrxA-ApSerpin-FA72 induced under IPTG conditions.

Fig. 2 shows the result of expression optimization of recombinant protein TrxA-ApSerpin-FA72.

In the figure, 1 is the pET-32a-ApSerpin-FA72/BL21 pre-treatment group, 2-6 are the pET-32a-ApSerpin-FA72/BL21 post-treatment groups, the treatment methods are 0.2 mmol/L IPTG, 0.4 mmol/L, respectively. L IPTG, 0.6 mmol/LIPTG, 0.8 mmol/L IPTG, 1 mmol/L IPTG.

Figure 3 shows the result of purification of recombinant protein TrxA-ApSerpin-FA72 inclusion bodies.

In the figure, M is the standard molecular weight protein, 1 is pET-32a-ApSerpin-FA72/BL21 after induction, 2-3 is washing solution containing 1% TritonX-100Tris-HCl, 4-5 is containing 2% TritonX-100Tris-HCl Washing solution, 6-7 is 2mol/L urea washing solution, 8 is purified inclusion body.

Figure 4 shows the results of screening the renaturation conditions of the recombinant protein TrxA-ApSerpin-FA72 inclusion bodies.

In the figure, A is Tris-HCl renaturation solution, 5% glycerol treatment group, B is Tris-HCl renaturation solution 0.4mol/LL-arginine treatment group, C is Tris-HCl renaturation solution, 0.9mmol/L Reduced glutathione, 0.2mmol/L oxidized glutathione treatment group, D is Tris-HCl renaturation solution, 5% Glycerol, 0.4mol/L L-arginine, 0.9mmol/L reduced glutathione GSH, 0.2mmol/L oxidized glutathione treatment group.

Figure 5 shows the results of SDS-PAGE detection of recombinant protein TrxA-ApSerpin-FA72 inclusion bodies under renaturation conditions.

In the figure, M is the standard molecular weight protein, 1 is Tris-HCl renaturation solution 5% Glycerol and 0.4mol/L L-arginine and 0.9mmol/L reduced glutathione and 0.2mmol/L oxidized glutathione Glutathione treatment group, 2 is Tris-HCl renaturing solution and 0.9mmol/L reduced glutathione and 0.2 mmol/L oxidized glutathione treatment group, 3 is Tris-HCl renaturing solution 0.4mol/LL -Arginine treatment group, 4 is Tris-HCl renaturation solution and 5% glycerol treatment group.

Figure 6 is a graph showing the results of the activity assay of trypsin after incubation with the renatured protein. In the figure, A is the control group of trypsin and renaturation solution incubation, B is the guanidine hydrochloride dilution and renaturation protein group, C is the guanidine hydrochloride dialysis renaturation protein group, D is the urea dilution and renaturation protein group, and E is the urea dialysis Refolding protein group, F is the renaturing protein group diluted with 2mol/L urea washing solution.

Figure 7 shows the results of incubation of trypsin with different concentrations of TrxA-ApSerpin-FA72.

Figure 8 is a graph showing the effect of pH on the activity of TrxA-ApSerpin-FA72.

Figure 9 is a graph showing the effect of temperature on the activity of TrxA-ApSerpin-FA72.

Detailed ways

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the following examples.

The materials in the present invention are: protein Marker (26614), reduced glutathione GSH, oxidized glutathione GSSG, guanidine hydrochloride, dithiothreitol DTT, L-Arg purchased from Peng Shida Experiment Co., Ltd.; Urea was purchased from Shanghai Sangon Bioengineering Company; trypsin (T1426) and benzoyl-L-arginyl-p-nitroaniline (B4875) were purchased from Sichuan Chenma Company; protein electrophoresis instrument was a product of Bio-Rad Company, The rest of the reagents are of domestic analytical grade. The reagents and materials can be purchased through public channels, and the equipment and instruments used in the process are common equipment in the art.

The strain pET32a-ApSerpin-FA72/BL21 used in the present invention has been disclosed in the prior art, and the existing patent CN109251936A has also disclosed the preparation of a fusion protein TrxA-ApSerpin-FA72 of a smooth turtle methaserine protease inhibitor Methods, the constructed engineering strain pET32a-ApSerpin-FA72/BL21 has become a well-known strain.

All materials, reagents and instruments selected in the present invention are well known in the art, but do not limit the implementation of the present invention, and some other reagents and equipment well known in the art are applicable to the implementation of the following embodiments of the present invention.

The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Modifications or substitutions made to the methods, steps or conditions of the present invention without departing from the spirit and essence of the present invention all belong to the scope of the present invention.

Example 1: A method for significantly improving the renaturation and activity of the fusion protein TrxA-ApSerpin-FA72 inclusion body protein of T. glabrata

The method for improving the renaturation and activity of the fusion protein TrxA-ApSerpin-FA72 inclusion body protein of the soft-shelled turtle methyl-serine protease inhibitor, the specific method steps are as follows:

(2) Transform the recombinant plasmid pET32a-ApSerpin-FA72 into Escherichia coli strain BL21 (DE3), screen positive single clones and culture, overnight at 37°C and 220r/min, and inoculate in LB medium at 1:100 the next day Medium (Amp ⁺ ) was cultured for 4h, the OD value of the bacteria was 0.6, added with different final concentrations of IPTG, induced at 32°C, 180r/min for 4h, centrifuged at 8000 × g for 10min to collect the precipitate, and resuspended the bacteria with phosphate buffer;

(2) Wash the precipitate with the prepared Tris-HCl buffer, fully dissolve the precipitate in the solution by ultrasonication for 2 min, centrifuge at 8000 × g for 10 min, repeat three times and wash the washing liquid remaining on the cells with Tris-HCl buffer net;

(3) The inclusion bodies were fully dissolved in the denaturing solution at 4°C for 1 h, centrifuged at 8000 × g for 15 min, and the renaturing buffer was slowly added to the lysing solution with a peristaltic pump within 18 h, and slowly stirred with a magnetic stirrer.

The phosphate buffer solution is prepared by mixing 137 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L Na ₂ HPO ₄ and 2 mmol/L KH ₂ PO ₄ .

Said, the prepared Tris-HCl buffer is prepared by mixing 2mol/L urea, 50mmol/L Tris-HCl, 100mmol/LNaCl, 0.1mmol/LDTT, and 0.1mmol/L EDTA.

Described, denaturation solution is 50mmol/L Tris-HCl, 100mmol/L NaCl, 0.1mmol/L DTT, 0.1mmol/L EDTA, 6mol/L guanidine hydrochloride, 0.4mol/L L-Arg mixes and makes, pH= 8.0.

The renaturation buffer is 50 mmol/L Tris-HCl, 100 mmol/L NaCl, 0.1 mmol/LDTT, 0.1 mmol/LEDTA, and is prepared by mixing.

Example 2: Expression of TrxA-ApSerpin-FA72 fusion protein and identification of expression form

Based on Example 1, the recombinant plasmid pET32a-ApSerpin-FA72 constructed in the laboratory was transformed into Escherichia coli strain BL21 (DE3), and positive single clones were screened, and cultured overnight at 37°C and 220 r/min, followed by 1:100 the next day. The bacteria were cultured in LB medium (Amp ⁺ ) for 4 hours, the OD value of the bacteria was 0.6, added with different final concentrations of IPTG, induced for 4 hours at 32 °C, 180 r/min, and centrifuged at 8000 × g for 10 min to collect the precipitate, and phosphate buffer was used. After the bacteria were resuspended, sonicated, the precipitate and supernatant were collected, and 12% SDS-PAGE was used to detect the expression of fusion protein and compare the protein induced by different concentrations of IPTG. The induction results are shown in FIG. 1 . After IPTG-induced expression of pET32a-ApSerpin-FA72/BL21, compared with before induction, a target band appeared at about 58 kD, as shown by the arrow A in FIG. 1 . The bacterial solution was ultrasonically broken, centrifuged to obtain the precipitate and supernatant, and the expression form of the recombinant protein was analyzed in 12% SDS-PAGE, and the obtained fusion protein was subjected to 12% SDS-PAGE gel electrophoresis. The band gel was blocked by transfer membrane, diluted goat anti-human IgGFc (HRP) antibody with blocking solution according to 1:5000, put the transfer membrane in it, incubated for 2h at room temperature, took out and washed 3 times with TBST solution, each wash 10min. Color photography. The results are shown in Fig. 1, B, showing that the recombinant protein TrxA-ApSerpin-FA72 mainly exists in the form of inclusion bodies. After Western blotting analysis, as shown in C in Figure 1, it was found that the recombinant protein was recognized by His-tag antibody with a specific band, indicating that the expressed protein was the target protein. As shown in Figure 2, the protein expression level was the highest in IPTG at 0.4 mmol/L.

Example 3: Isolation and purification of inclusion bodies of TrxA-ApSerpin-FA72 fusion protein

Based on the preparation methods in Example 1 and Example 2, the precipitate was washed with Tris-HCl buffer solution containing 1%, 2% TritonX-100 and 2mol/L urea, and the precipitate in the solution was fully dissolved by ultrasonication for 2 min, 8000× g centrifugation for 10 min, and after repeating three times, the washing solution remaining on the cells was washed with Tris-HCl buffer. Detergents with different concentrations of ingredients removed the non-target bands in the precipitate, and finally the bacterial cells were released to obtain a large number of relatively pure inclusion bodies. The inclusion bodies of TrxA-ApSerpin-FA72 fusion protein were significantly higher than those of other buffer-treated groups.

Example 4: Screening of renaturation conditions for recombinant protein TrxA-ApSerpin-FA72

Based on the above series of examples, the inclusion bodies were fully dissolved in the denaturing solution at 4°C for 1 h, centrifuged at 8000 × g for 15 min, and the different renaturing buffer treatment groups were slowly added to the lysing solution with a peristaltic pump within 18 h, and a magnetic stirrer was used. Stir slowly, in addition to 50mmol/L Tris-HCl, 100mmol/L NaCl, 0.1mmol/L DTT, 0.1mmol/L EDTA, the renaturation buffers of different treatment groups also contain 0.4mol/L L-Arg, 0.9 mmol/L GSH, 0.2 mmol/L GSSG, 5% glycerol, the absorbance at OD320 of each treatment group was detected as a measure of light scattering, insoluble proteins had absorption peaks at _OD320 , and soluble proteins had no absorption peaks at 320nm. The renaturation efficiency of the same denatured protein solution in the renaturation solution of different components, the denaturation solution without inclusion bodies was used as the control, and the turbidity was corrected. Measure the changes of absorbance at 2h, 14h and 26h, and screen out the optimal renaturation conditions. As shown in accompanying drawing 4, add the OD of redox system (50mmol/L Tris-HCl, 100mmol/L NaCl, 0.1mmol/L DTT, 0.1mmol/L EDTA, 0.9mmol/L GSH, 0.2mmol/L GSSG) ₃₂₀ grows the fastest. During the whole renaturation process, the OD ₃₂₀ of the renaturing solution containing L-Arg grows slowly, the protein does not absorb at 320nm, and the value is low, so the effect of three different substances in the renaturation process L-Arg>glycerol>redox system (GSH, GSSG). The renatured protein was verified by SDS-PAGE, see Figure 5, and the results were consistent with Figure 4.

Example 5: Determination of the content of recombinant protein TrxA-ApSerpin-FA72

Based on the above example, by Bradford method, with bovine serum albumin as the standard of 1 mg/mL, the protein was diluted with different concentration gradients of 0, 0.1, 0.3, 0.5, 0.7, 1.0 mg/mL to make a standard curve to measure the protein concentration. 1-10 μg/mL.

Example 6: Activity detection of recombinant protein TrxA-ApSerpin-FA72

Based on the above example, the activity of TrxA-ApSerpin-FA72 was determined, that is, the inhibitory activity to trypsin was determined. According to the experimental method of Sigma Company and the According to the method reported by TangY, NegiP, JohnsonET and others, trypsin was dissolved in 0.1mmol/L HCl at a concentration of 0.2mg/mL, and benzoyl-L-arginyl-p-nitroaniline (B4875) was dissolved in buffer 50mmol /L Tris-HCl, 120mmol/L CaCl ₂ , 100mmol/L NaCl, pH=8.0, take 50μL of enzyme solution and 50μL of different concentrations of serine protease inhibitors and incubate at 37°C for 30min, add 100μL of B4875 (1mg/mL) . The microplate reader continuously detects the change of OD ₄₀₅ absorbance for 15 minutes, and defines the activity unit of nitroaniline: a change of 0.01 in OD ₄₀₅ is an activity unit.

Example 7: Comparison of renaturation methods for recombinant protein TrxA-ApSerpin-FA72

According to the above-mentioned series of embodiments, according to the renaturation method and the denaturing liquid composition, different renaturation processes are carried out, guanidine hydrochloride dilution renaturation, guanidine hydrochloride dialysis renaturation, urea dilution renaturation, urea dialysis renaturation (wherein 2mol/L The urea eluate was subjected to dialysis renaturation of urea), and the inhibitory activity of trypsin and the IC ₅₀ of trypsin inhibition by different conditions of renatured proteins were determined. The test results are shown in Table 1. Comparing the renaturation of urea dialysis, urea dilution renaturation, dialysis renaturation of guanidine hydrochloride, and guanidine hydrochloride dilution renaturation, the denatured protein solution of the same concentration was renatured by different methods, and the amount of soluble protein obtained. However, under the same volume, the soluble protein concentration of guanidine hydrochloride dilution and renaturation was significantly lower than other renaturation methods. As shown in Figure 6, after incubation of different proteins with trypsin for 30 min, the increase in OD ₄₀₅ was small within the same time period, the less p-nitroaniline was generated, and the stronger the inhibitory activity was. The protein TrxA-ApSerpin-FA72 has a strong inhibitory effect on trypsin.

Table 1: Soluble protein concentrations under different renaturation methods

renaturation mode OD_595nm Concentration (mg/mL) Diluted renaturation with guanidine hydrochloride 0.899 0.1688 Guanidine hydrochloride renaturation by dialysis 1.425 0.6422 Urea dilution and renaturation 0.838 0.1214 urea dialysis renaturation 1.194 0.4372 2mol/L urea eluate 1.122 0.3733

The inhibitory activity of the renatured protein TrxA-ApSerpin-FA72 on trypsin was further detected. After incubation with different concentrations of the recombinant protein TrxA-ApSerpin-FA72 and trypsin for 30 min, benzoyl-L-arginyl-p-nitroanilide was added. The inhibitory activity of TrxA-ApSerpin-FA72 on trypsin was detected. As shown in Fig. 7, as the concentration of TrxA-ApSerpin-FA72 increased, the OD ₄₀₅ per unit time increased slowly, and the formation rate of p-nitroaniline decreased. The results showed that the renatured recombinant protein TrxA-ApSerpin-FA72 had a concentration-dependent inhibitory effect on trypsin. The IC ₅₀ of recombinant protein TrxA-ApSerpin-FA72 was 4.8mmol/L.

Example 8: Optimum pH of TrxA-ApSerpin-FA72 fusion protein

Based on the above series of examples, the effect of TrxA-ApSerpin-FA72 on the activity of trypsin in the pH range of 5-11 was studied, and the pH with the highest activity was determined to be the optimal pH in the experiment. The renatured TrxA-ApSerpin-FA72 was incubated with trypsin at different pH at 30°C for 30min, and the optimal pH was calculated. The test results are shown in Figure 8. After the renatured recombinant protein TrxA-ApSerpin-FA72 was freeze-dried, it was dissolved in solutions at different pH, and the inhibitory activity of the recombinant protein TrxA-ApSerpin-FA72 to trypsin was determined. Serine protease inhibitors have high inhibitory activity between pH=7-9, and the stability of inhibitors is poor outside this pH range.

Example 9: Thermostability of recombinant protein TrxA-ApSerpin-FA72

Based on the above series of examples, the inhibitory effect of temperature on trypsin was studied. The inhibitory activity of TrxA-ApSerpin-FA72 was detected at different temperatures from 20°C to 70°C. Trypsin was incubated with the inhibitor for 30min and 2h. The two groups of experiments have the highest inhibitory activity. The temperature was set as the optimal temperature for trypsin inhibitor. The assay results are shown in Figure 9. The inhibitory activity of recombinant protein TrxA-ApSerpin-FA72 on trypsin is in the temperature range of 20 to 70 °C. TrxA-ApSerpin-FA72 and trypsin have the same results at different incubation times. When the protease temperature was above 30℃, the relative residual activity of TrxA-ApSerpin-FA72 increased with the increase of temperature and reached the highest at 60℃, after which the activity decreased significantly and showed no inhibitory activity at 70℃.

As described above, the present invention can be well realized. The above-mentioned embodiments are only to describe the preferred embodiments of the present invention, and do not limit the scope of the present invention. Various changes and improvements made by technical personnel to the technical solutions of the present invention shall fall within the protection scope determined by the present invention.

Claims (6)

1. A method for improving the renaturation and activity of the fusion protein TrxA -ApSerpin- FA72 inclusion body protein of the smooth soft-shelled turtle methylserine protease inhibitor, the specific method steps are as follows: The recombinant plasmid pET32a -ApSerpin- FA72 was transformed into Escherichia coli strain BL21(DE3), and positive single clones were screened for cultivation, and cultured overnight at 37°C and 220r/min. The next day, the bacteria were inoculated in LB medium (Amp 1:100). ⁺ ) cultured for 4 hours, the OD value of the bacteria was 0.6, added IPTG with different final concentrations, induced for 4 hours at 32 °C, 180 r/min, centrifuged at 8000 × g for 10 minutes to collect the precipitate, and resuspended the bacteria with phosphate buffer; (2) Wash the precipitate with the prepared Tris-HCl buffer, sonicate for 2 min to fully dissolve the precipitate in the solution, centrifuge at 8000 × g for 10 min, repeat three times and wash the remaining washing solution with Tris-HCl buffer. net; (3) The inclusion bodies were fully dissolved in the denaturing solution at 4°C for 1 h, centrifuged at 8000 × g for 15 min, and the renaturation buffer was slowly added to the lysing solution with a peristaltic pump within 18 h, and slowly stirred with a magnetic stirrer. 2 . The method for improving the renaturation and activity of inclusion body protein of T. glabrata merpin fusion protein TrxA- ApSerpin -FA72 as claimed in claim 1 , wherein the IPTG concentration is 0.4mmol/L. 3 . 3. the method for improving the fusion protein TrxA -ApSerpin- FA72 inclusion body protein and the activity of the smooth turtle methyl-serine protease inhibitor fusion protein as claimed in claim 1, is characterized in that, phosphate buffer is 137mmol/L NaCl, 2.7mmol/ It is prepared by mixing L KCl, 10mmol/L Na ₂ HPO ₄ and 2mmol/L KH ₂ PO ₄ . 4. the method for improving the fusion protein TrxA -ApSerpin- FA72 inclusion body protein of smooth soft-shelled turtle as claimed in claim 1 and the activity, it is characterised in that the Tris-HCl buffer of preparation is 2mol/L urea , 50mmol/L Tris-HCl, 100mmol/L NaCl, 0.1mmol/L DTT, 0.1mmol/L EDTA were mixed. 5. the method for improving smooth turtle methylserine protease inhibitor fusion protein TrxA -ApSerpin- FA72 inclusion body protein renaturation and activity as claimed in claim 1, is characterized in that, denaturant is 50mmol/L Tris-HCl, 100mmol/ It is prepared by mixing LNaCl, 0.1mmol/L DTT, 0.1mmol/L EDTA, 6mol/L guanidine hydrochloride, and 0.4mol/L L-Arg, pH=8.0. 6. the method for improving the fusion protein TrxA -ApSerpin- FA72 inclusion body protein of smooth soft-shelled turtle as claimed in claim 1 and the activity, it is characterized in that, the renaturation buffer is 50mmol/L Tris-HCl, 100mmol/L NaCl, 0.1mmol/L DTT, 0.1mmol/L EDTA, mixed to prepare.