Method for secretory expression of recombinant mechanical structural protein by using escherichia coli and application of recombinant mechanical structural protein

Abstract

The invention discloses a method for secretory expression of recombinant mechanical structural protein by using escherichia coli and application, and relates to the field of bioengineering. According to the method, signal peptides are introduced to the N end of recombinant mechanical structural proteins, and the signal peptides, namely OmpA, PelB, PhoA, DsbA and STII, capable of guiding different recombinant mechanical structural proteins to secrete and express are screened from six natural signal peptides from escherichia coli and other species. Meanwhile, the yield of extracellular protein is further improved by combining an amino acid tRNA supplement strategy. According to the method, the recombinant escherichia coli strain capable of efficiently secreting and expressing the mechanical structure protein can be obtained, the downstream purification process is simplified, and the method has important application value.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a method and application of using Escherichia coli to secrete and express recombinant mechanical structural proteins. Background Technology

[0002] Natural mechanical structural proteins and materials derived from spider silk, elastin, collagen, etc., possess properties such as high mechanical strength, biodegradability, and low immunogenicity, thus finding increasing applications in fields such as biomedicine, military industry, and aerospace. Based on these natural proteins, modifying or designing high-performance mechanical structural proteins and utilizing microorganisms for efficient production can overcome the limitations of mass production difficulties and unstable quality of natural proteins, potentially enabling the widespread application of high-performance mechanical structural proteins.

[0003] Escherichia coli, as one of the best-suited hosts, has advantages such as short growth cycle, low culture cost, and suitability for industrial production, and is now widely used as a cell factory for producing heterologous proteins. Currently, research on using E. coli as a host to express repetitive mechanical structural proteins has made some progress in the development and screening of expression elements and the optimization of expression hosts. However, there are still some problems to be solved in the current use of microorganisms to express high-performance mechanical structural proteins, mainly manifested in difficulties in protein secretion and low expression levels of large molecular weight proteins. Some reports have described how E. coli uses signal peptides to guide the secretion of different proteins into the periplasm or culture medium, but these proteins generally have small molecular weights. There are currently no precedents for E. coli secreting and expressing large molecular weight, highly repetitive mechanical structural proteins. Furthermore, signal peptides are not universally applicable to different proteins; different types of signal peptides have different guiding efficiencies for the same protein, and the optimal signal peptide for one protein expression may not be suitable for another. Therefore, it is necessary to screen for signal peptides from different sources for E. coli secreted mechanical structural proteins to meet the needs of exogenous repetitive mechanical structural protein expression and secretion. At the same time, it is also necessary to modify the host to further increase protein production, taking into account the characteristics of the simple amino acid composition and high repetitiveness of such proteins. Summary of the Invention

[0004] The purpose of this invention is to provide a method and application for expressing recombinant mechanical structural proteins using Escherichia coli secretion, in order to solve the problems of difficulty in secreting and low expression levels of recombinant mechanical structural proteins in Escherichia coli. This strategy can improve the secretion level of mechanical structural proteins and has important industrial application value.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] A method and application of expressing recombinant mechanical structural proteins using Escherichia coli secretion, wherein the method is obtained by introducing a signal peptide at the N-terminus of the expressed recombinant protein fragment.

[0007] In one or more embodiments, the recombinant mechanical structural protein includes SRT-ELP36 shown in SEQ ID NO.7, SRT-ELP24 shown in SEQ ID NO.8, and ELP-K72 shown in SEQ ID NO.9.

[0008] In one or more embodiments, the signal peptide includes, but is not limited to, OmpA as shown in SEQ ID NO.1, PelB as shown in SEQ ID NO.2, PhoA as shown in SEQ ID NO.3, Cel-CD as shown in SEQ ID NO.4, DsbA as shown in SEQ ID NO.5, and STⅡ as shown in SEQ ID NO.6.

[0009] In one or more embodiments, the signal peptide, comprising OmpA (SEQ ID NO.1), PelB (SEQ ID NO.2), PhoA (SEQ ID NO.3), DsbA (SEQ ID NO.5), and STⅡ (SEQ ID NO.6), can guide the secretion of mechanical structural proteins extracellularly. Preferably, the signal peptide has different secretion effects on different mechanical structural proteins; for SRT-ELP36, it is OmpA (SEQ ID NO.1); for SRT-ELP24, it is DsbA (SEQ ID NO.5); and for SRT-ELP36, it is OmpA (SEQ ID NO.1).

[0010] In one or more embodiments, the recombinant mechanical structural protein is expressed using pET25b as the expression vector and Escherichia coli BL21(DE3) as the host.

[0011] In another aspect of the invention, a recombinant Escherichia coli strain overexpressing an amino acid tRNA gene is provided. This system is a dual plasmid expression system, comprising: (a) an Escherichia coli signal peptide-mechanical structure protein expression cassette, and (b) an expression cassette for the following protein: glyVXY of the amino acid sequence shown in SEQ ID NO.10.

[0012] In one or more embodiments, (b) further includes an expression cassette introducing a protein selected from the group consisting of valU of the amino acid sequence shown in SEQ ID NO. 11 or proL of the amino acid sequence shown in SEQ ID NO. 12. Further, the engineered bacteria are supplemented with amino acid tRNA to further enhance the extracellular production of the mechanical structural protein.

[0013] The present invention discloses the following technical effects:

[0014] By introducing a signal peptide at the N-terminus of a recombinant protein fragment, recombinant mechanical structural proteins containing the signal peptide are obtained. Screening of six signal peptides revealed that OmpA, PelB, PhoA, DsbA, and STⅡ can guide the secretory expression of different recombinant mechanical structural proteins. Furthermore, a tRNA supplementation strategy can be combined to further enhance extracellular protein production. This invention enables efficient secretory expression of mechanical structural proteins using *E. coli*, simplifying downstream separation and purification processes, and has significant application value.

[0015] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0016] Figure 1 ---- Schematic diagram of a mechanical structural protein expression vector incorporating different signal peptides.

[0017] Figure 2 ----Escherichia coli BL21(DE3) fused with different signal peptides secreted and expressed recombinant protein SRT-ELP36. (a. Growth curves of recombinant bacteria containing different signal peptides S36; b. SDS-PAGE and Western Blot analysis of intracellular protein expression levels in different S36 recombinant bacteria; c. SDS-PAGE and Western Blot analysis of extracellular protein secretion levels in different S36 recombinant bacteria.)

[0018] Figure 3 ----Escherichia coli BL21(DE3) fused with different signal peptides secreted and expressed recombinant protein SRT-ELP24. (a. Growth curves of recombinant bacteria containing different signal peptides S24; b. SDS-PAGE and Western Blot analysis of intracellular protein expression levels in different S24 recombinant bacteria; c. SDS-PAGE and Western Blot analysis of extracellular protein secretion levels in different S24 recombinant bacteria.)

[0019] Figure 4 ----Escherichia coli BL21(DE3) fused with different signal peptides secreted and expressed recombinant protein ELP-K72. (a. Growth curves of recombinant bacteria containing different signal peptides K72; b. SDS-PAGE and Western Blot analysis of intracellular protein expression levels in recombinant bacteria containing different K72; c. SDS-PAGE and Western Blot analysis of extracellular protein secretion levels in recombinant bacteria containing different K72.)

[0020] Figure 5----Analysis of tRNA protein expression in recombinant strain OmpA36 supplemented with different amino acid tRNAs. (a. Growth curves of recombinant OmpA36 strains supplemented with different amino acid tRNAs; b. SDS-PAGE analysis of intracellular protein expression levels in recombinant OmpA36 strains supplemented with different amino acid tRNAs; c. SDS-PAGE analysis of extracellular protein secretion levels in recombinant OmpA36 strains supplemented with different amino acid tRNAs.)

[0021] Figure 6 ----Analysis of tRNA protein expression in recombinant strain DsbA24 supplemented with different amino acid tRNAs. (a. Growth curves of recombinant DsbA24 strains supplemented with different amino acid tRNAs; b. SDS-PAGE analysis of intracellular protein expression levels in recombinant DsbA24 strains supplemented with different amino acid tRNAs; c. SDS-PAGE analysis of extracellular protein secretion levels in recombinant DsbA24 strains supplemented with different amino acid tRNAs.)

[0022] Figure 7 ----Analysis of tRNA protein expression in recombinant OmpA72 strain supplemented with different amino acid tRNAs. (a. Growth curves of OmpA72 recombinant bacteria supplemented with different amino acid tRNAs; b. SDS-PAGE analysis of intracellular protein expression levels in OmpA72 recombinant bacteria supplemented with different amino acid tRNAs; c. SDS-PAGE analysis of extracellular protein secretion levels in OmpA72 recombinant bacteria supplemented with different amino acid tRNAs.) Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] Unless otherwise specified, the technical means used in the following examples are conventional means and commercially available instruments and reagents that are well known to those skilled in the art. For reference, please refer to "Molecular Cloning: A Laboratory Manual (3rd Edition)" (Science Press), "Microbiology Experiments (4th Edition)" (Higher Education Press), and the manufacturer's instructions for the corresponding instruments and reagents.

[0029] Related materials and methods in this invention

[0030] 1. Candidate signal peptide-related amino acid sequence, target protein amino acid sequence, and amino acid-tRNA nucleotide sequence

[0031] The relevant signal peptide amino acid sequence, target protein amino acid sequence, and amino acid tRNA nucleotide sequence involved in this invention are shown in Table 1.

[0032] Table 1

[0033]

[0034]

[0035] 2. Plasmids and strains

[0036] The relevant plasmids involved in this invention are shown in Table 2.

[0037] Table 2

[0038]

[0039] Similarly, recombinant plasmids containing the mechanical structural proteins SRT-ELP24 and ELP-K72 were constructed using the same method.

[0040] [1]LI Y, LI J, SUN J, et al. Bioinspired and Mechanically Strong FibersBased on Engineered Non-Spider Chimeric Proteins[J]. Angewandte ChemieInternational Edition, 2020, 59(21):8148-52.

[0041] The relevant strains involved in this invention are shown in Table 3.

[0042] Table 3

[0043]

[0044] Similarly, recombinant bacteria containing the mechanical structural proteins SRT-ELP24 and ELP-K72 were constructed using the same method.

[0045] [1]LI Y, LI J, SUN J, et al. Bioinspired and Mechanically Strong FibersBased on Engineered Non-Spider Chimeric Proteins[J]. Angewandte ChemieInternational Edition, 2020, 59(21):8148-52.

[0046] 3. Primers

[0047] The relevant primer sequences involved in this invention are shown in Table 4.

[0048] Table 4

[0049]

[0050]

[0051] *The underlined sequence indicates the enzyme cleavage site.

[0052] 4. Culture medium

[0053] (1) LB medium

[0054] Yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L. Add 2% agar powder to the solid culture medium. Add appropriate concentrations of antibiotics as needed.

[0055] (2) TB culture medium

[0056] Yeast extract 24 g / L, peptone 12 g / L, glycerol 4 mL / L, KH2PO4 2.32 g / L, K2HPO4 12.54 g / L.

[0057] 5. Molecular biology methods

[0058] Conventional molecular biology techniques, including small-scale plasmid extraction, DNA gel cutting and recovery, and DNA fragment purification, are all performed in accordance with the instructions of the relevant kits.

[0059] (1) The Q5 high-fidelity DNA polymerase PCR reaction system is shown in Table 5:

[0060] Table 5

[0061] Element volume 10μM Forward Primer 2.5μL 10μM Reverse Primer 2.5μL DNA template 0.2μL Q5 High-Fidelity 2×Master Mix 25μL sterile deionized water Make up to 50 μL

[0062] (2) The PCR reaction conditions are shown in Table 6:

[0063] Table 6

[0064]

[0065] (3) The enzyme digestion reaction system is shown in Table 7:

[0066] Table 7

[0067] plasmid DNA PCR products 10×Buffer 5μL 5μL Enzyme 1 1μL 1μL enzyme 2 1μL 1μL DNA ≤1μg ≤0.2μg <![CDATA[ddH2O]]> Up to 50μL Up to 50μL Total volume 50μL 50μL

[0068] *If the reaction temperatures of the enzymes are different, add the corresponding restriction enzymes in order from low temperature to high temperature to carry out stepwise enzymatic digestion reactions.

[0069] (4) The connection reaction system is as follows:

[0070] Prepare a 5-10 μL DNA solution by mixing the vector (50 ng, 25 fmol), plasmid vector, and insert DNA fragment (25-250 fmol) (a plasmid vector: insert DNA fragment ratio of approximately 1:6 is preferred). Add an equal volume (5-10 μL) of Ligation Mix (TaKaRa) to the DNA solution and mix thoroughly; incubate at 16°C for 30 minutes.

[0071] Example 1: Screening signal peptides for secretory expression of the mechanical structural protein SRT-ELP36

[0072] Choosing the right signal peptide is crucial for achieving and improving protein secretion efficiency, but signal peptides lack universal applicability to different proteins. The optimal signal peptide for one protein expression may not be suitable for another; different types of signal peptides also have varying guiding efficiencies for the same protein. Currently, there are no reports on solving the problem of secreting large-molecule, highly reproducible mechanical structural proteins in *E. coli*. Therefore, it is necessary to screen for signal peptides from different sources for secreting mechanical structural proteins in *E. coli* to meet the needs of protein expression and secretion.

[0073] Using plasmid pETS36, which expresses the mechanical structural protein SRT-ELP36, as a template, PCR amplification was performed using primers pET25b-F (containing the EcoRI restriction site) and pET25b-R (containing the BamHI restriction site) to obtain the expression vector fragment. After the PCR reaction, the PCR products were detected by gel electrophoresis. When the band size was correct and there were no impurities, a purification kit was used to remove enzymes and salt ions from the PCR products. Restriction endonucleases EcoRI and BamHI were used for digestion; plasmid pETS36 was digested with restriction endonucleases NdeI and EcoRI, and the digested SRT-ELP36 fragment was recovered using a gel extraction kit. Signal peptides were synthesized according to the codon preference of *E. coli*, and BamHI and NdeI restriction sites were introduced upstream and downstream of each peptide for simultaneous digestion. A purification kit was used to remove enzymes and salt ions from the PCR products.

[0074] Using TaKaRa Ligation Mix, the above-mentioned vector pET25b, the mechanical structural protein SRT-ELP36, and the signal peptide gene fragment were ligated to obtain circular plasmids. The ligation product was transformed into *E. coli* BL21(DE3), and after screening with ampicillin (Amp), single colonies were picked, amplified, and plasmids were extracted. After double digestion with BamHI and EcoRI, sequencing yielded recombinant plasmids pOmS36, pPeS36, pPhS36, pCeS36, pDsS36, and pSTS36 (fusion protein structures shown below). Figure 1 (As shown).

[0075] Example 2: Protein Expression Analysis of Recombinant S36 Bacteria Containing Different Signal Peptides

[0076] S36 recombinant bacteria containing different signal peptides were induced and cultured until OD 600 When the culture reaches approximately 4 hours, induction is performed using IPTG at a final concentration of 0.5 mM, and OD is measured every 2 hours. 600 And intracellular and extracellular protein expression levels. When detecting protein expression levels, the supernatant after centrifugation of the fermentation broth was used directly for the detection of extracellular proteins, and the bacterial cells were resuspended in PBS to OD0.05. 600 =5. Detect intracellular protein expression levels.

[0077] like Figure 2 As shown in Figure a, the growth of S36 recombinant bacteria containing different signal peptides differed from that of the S36 control group without signal peptides; most recombinant bacteria grew worse than S36, while the recombinant bacteria Cel-CD36 grew better than S36. Protein expression levels were detected using SDS-PAGE and Western spectroscopy. Figure 2 As shown in b and c, signal peptides OmpA, PelB, PhoA, DsbA, and STⅡ can guide SRT-ELP36 secretion into the culture supernatant, but the corresponding intracellular protein content is reduced. However, the signal peptide Cel-CD cannot guide protein secretion into the extracellular space, and the intracellular protein expression level is not significantly different from the control group without the signal peptide. Therefore, through screening and verification of signal peptides, recombinant E. coli strains capable of guiding the secretion of mechanical structural proteins into the extracellular space were successfully obtained.

[0078] Example 3: Analysis of the results of screening signal peptides for the secretion and expression of the mechanical structural protein SRT-ELP24.

[0079] To screen for signal peptides for the secretory expression of the mechanical structural protein SRT-ELP24, the construction steps of the expression vector and recombinant bacteria were basically the same as in Examples 1 and 2, except that the target protein was different; SRT-ELP36 was replaced with SRT-ELP24. Figure 3 As shown in b and c, signal peptides OmpA, PelB, PhoA, DsbA, and STII can also guide the secretion of SRT-ELP24 into the culture medium supernatant.

[0080] Example 4: Analysis of the results of screening signal peptides for the secretory expression of the mechanical structural protein ELP-K72

[0081] To screen for signal peptides for the secretory expression of the mechanical structural protein ELP-K72, the construction steps of the expression vector and recombinant bacteria were basically the same as in Examples 1 and 2, except that the target protein was different; SRT-ELP36 was replaced with ELP-K72. Figure 4 As shown in b and c, signal peptides OmpA, PelB, PhoA, DsbA, and STII can also guide ELP-K72 to be secreted into the culture medium supernatant, and the secretion amount is relatively high.

[0082] Example 4: Supplementing amino acid tRNA promotes protein expression in recombinant bacteria OmpA36.

[0083] Recent research has shown that a coordinated balance between recombinant protein translation and transport rates is key to improving the secretion efficiency of *E. coli*. Therefore, after achieving secretory expression of recombinant mechanical structural proteins in *E. coli*, it is necessary to simultaneously improve the host's translational capabilities to further increase protein yield, taking advantage of the protein's simple and highly repetitive amino acid composition. Because the recombinant protein SRT-ELP36 (sequence PAATAVSHTTHHAP-(VPGKG)5VPGVG) is a highly repetitive sequence containing only a few amino acids, with glycine, valine, and proline being predominantly present, a large amount of corresponding tRNA is required during protein translation. Furthermore, the demand for target amino acids increases with the number of tandem sequences, making the overexpression of large molecular weight recombinant proteins exceptionally difficult. This invention aims to verify whether supplementing with different amino acid tRNAs promotes protein secretion expression. The proposed approach is to increase the amount of tRNAgly, tRNAval, and tRNApro genes to increase the amount of corresponding amino acid tRNAs, thereby increasing the accumulation of aminoacyl-tRNAs and promoting the translation of the target protein. This invention aims to increase the supply of tRNA for the corresponding amino acids by increasing the copy number of the genes for glycine, valine, and proline tRNA, thereby improving protein expression levels.

[0084] Using *E. coli* BL21(DE3) as a template, PCR amplification was performed using primers glyVXY-F (containing EcoRIV) and glyVXY-R (containing BamHI), valU-F (containing BamHI) and valU-R (containing SphHI), and proL-F (containing SphHI) and proL-R (containing SalHI) to obtain the corresponding tRNA gene fragments glyVXY, valU, and proL. After the PCR reaction, the PCR products were detected by gel electrophoresis. When the bands were of the correct size and free of impurities, a purification kit was used to remove enzymes and salt ions from the PCR products. Restriction endonucleases were then used for digestion; simultaneously, plasmid pACYC184 was digested with restriction endonucleases, and the digested vector fragments were recovered using a gel extraction kit. Using TaKaRa Ligation Mix, the above vector pACYC184 and the tRNA gene fragments were ligated to obtain a circular plasmid. The ligation product was transformed into *E. coli* DH5α. After screening with chloramphenicol (Cm), single colonies were picked, and plasmids were amplified and extracted. These plasmids were identified by double digestion with EcoRI and SalI, and then sequenced to obtain recombinant plasmids pAglyVXY, pAvalU, and pAproL. The verified plasmids were transformed into recombinant bacteria OmpA36. After screening with ampicillin and chloramphenicol, recombinant bacteria OmpA36-gly, OmpA36-val, and OmpA36-pro were obtained.

[0085] Recombinant OmpA36 bacteria containing different tRNAs were induced and their protein expression was detected. The culture methods and protein expression detection levels were the same as in Example 2. Figure 5 As shown in Figure a, the growth of OmpA36 recombinant bacteria containing different tRNAs was not significantly different from that of recombinant OmpA36. Protein expression was detected using SDS-PAGE, such as... Figure 5 As shown in b and c, supplementing glycine and proline tRNA can increase the production of the extracellular protein SRT-ELP36 in recombinant bacteria OmpA36, while there is no significant difference in the production of intracellular protein.

[0086] Example 5: Supplementing amino acid tRNA promotes protein expression in recombinant bacteria DsbA24.

[0087] Example 5 shows the protein expression results of recombinant strain DsbA24 after supplementing it with amino acid tRNA. The construction of the recombinant strain, the strain culture method, and the protein expression detection method are basically the same as in Example 4, except that the target protein is different; protein SRT-ELP36 is replaced with SRT-ELP24. Figure 6 As shown in c, supplementing glycine and proline tRNA can increase the yield of the extracellular protein SRT-ELP24 of recombinant bacteria DsbA24.

[0088] Example 6: Supplementing amino acid tRNA promotes protein expression in recombinant bacteria OmpA72.

[0089] Example 6 shows the protein expression results of recombinant strain OmpA72 after supplementing it with amino acid tRNA. The construction of the recombinant strain, the strain culture method, and the protein expression detection method were basically the same as in Example 4, except that the target protein was different; SRT-ELP36 was replaced with ELP-K72. Because the amino acid composition of OmpA72 protein differs from that of OmpA36 and DsbA24, the results obtained when screening for supplemented tRNAs with different amino acids also differed. Figure 7 As shown in c, supplementing glycine tRNA alone can slightly increase the yield of extracellular protein ELP-K72 in recombinant bacteria OmpA72.

[0090] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the design concept of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method and application for expressing recombinant mechanical structural proteins using Escherichia coli secretion, characterized in that, The method is obtained by introducing a signal peptide at the N-terminus of the expressed recombinant protein fragment.

2. The method according to claim 1, characterized in that, The recombinant mechanical structural proteins include, but are not limited to, SRT-ELP36 shown in SEQ ID NO.7, SRT-ELP24 shown in SEQ ID NO.8, and ELP-K72 shown in SEQ ID NO.

9.

3. The method according to claim 1, characterized in that, The signal peptides include, but are not limited to, OmpA as shown in SEQ ID NO.1, PelB as shown in SEQ ID NO.2, PhoA as shown in SEQ ID NO.3, Cel-CD as shown in SEQ ID NO.4, DsbA as shown in SEQ ID NO.5, and STⅡ as shown in SEQ ID NO.

6. Preferably, the signal peptide has different secretion effects on different mechanical structural proteins; for SRT-ELP36 it is OmpA as shown in SEQ ID NO.1, for SRT-ELP24 it is DsbA as shown in SEQ ID NO.5, and for SRT-ELP36 it is OmpA as shown in SEQ ID NO.

1.

4. The method according to claim 1, characterized in that, pET25b was used as the expression vector, and Escherichia coli BL21(DE3) was used as the host.

5. A method and its application for increasing the secretory expression level of recombinant mechanical structural proteins using *Escherichia coli*, characterized in that... Different amino acid tRNAs were screened and supplemented to improve the secretory expression level of mechanical structural proteins.

6. The method according to claim 5, characterized in that, A recombinant Escherichia coli strain overexpressing an amino acid tRNA gene is provided. The system is a dual plasmid expression system, comprising: (a) an Escherichia coli signal peptide-mechanical structure protein expression cassette, and (b) an expression cassette for the following proteins: glyVXY of the amino acid sequence shown in SEQ ID NO.10, or valU of the amino acid sequence shown in SEQ ID NO.11, or proL of the amino acid sequence shown in SEQ ID NO.

12.

7. The application of Escherichia coli according to claim 5 or 6, for improving the level of recombinant mechanical structural protein secreted and expressed by Escherichia coli.

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