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Recombinant spider silk protein and application thereof

A technology of spidroin and protease, which is applied in the protein field, can solve the problems of ignoring the degradability of spidroin protein, achieve the effect of ensuring mechanical properties or biological quality, wide application, and improving application value

Active Publication Date: 2019-08-13
SUZHOU BAIYUAN GENT CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, researchers have not realized that spidroin proteins with high degradation performance and high degradation controllability have broader application value and market potential, and have completely ignored the improvement of the degradability of spidroin proteins. Someone proposed an effective method to improve the degradability of spidroin protein and make the degradability of spidroin protein controllable

Method used

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  • Recombinant spider silk protein and application thereof
  • Recombinant spider silk protein and application thereof
  • Recombinant spider silk protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Artificially synthesized recombinant spidroin gene MaSp1

[0050] The sequence structure of the recombinant spidroin protein selected in this embodiment is [T 1 -W n -T 2 -W n ] m , where n=1, m=4, T 1 The amino acid sequence of the spidroin domain monomer is shown in SEQ ID NO.1, T 2 The amino acid sequence of the spidroin protein domain monomer is shown in SEQ ID NO.2, the amino acid sequence of the protease cleavage site of W is KKKKSSSSSESSRSSSSSSS (the letters above are common amino acid abbreviations), and the recombinant spidroin protein The amino acid sequence is shown in SEQ ID NO.13. According to the recombinant spidroin protein sequence selected above and the principle of Escherichia coli preferred codon usage, a nucleotide sequence encoding a recombinant spidroin protein consistent with the amino acid sequence of the recombinant spidroin protein was designed , see the nucleotide sequence of SEQ ID NO.14 in the sequence listing.

Embodiment 2

[0051] Example 2. High expression of the gene MaSp1 of artificially synthesized recombinant spidroin in Escherichia coli

[0052] (1) The MaSp1 gene was synthesized by a gene synthesis company, cloned into the prokaryotic expression vector pET-28a+ containing a strong T7 promoter, and constructed into a recombinant plasmid pET-28a+-MaSp1 containing the artificially synthesized MaSp1 gene;

[0053] (2) Prepare Escherichia coli BL21(DE3) competent cells, and transform the recombinant plasmid pET-28a+-MaSp1 into the host cell BL21(DE3) by heat shock method (heat shock at 42°C for 45 seconds) to obtain the engineered plasmid containing the recombinant plasmid strains;

[0054] (3) Inoculate the engineered strain into the LB culture medium solution, culture it on a shaker at 37°C at 220rpm, when the concentration OD600 of the engineered strain reaches 0.6-0.8, add 0.5mmol / L IPTG, induce expression at 37°C for 6 hours, and then Lysis was carried out, and the resulting cell lysate w...

Embodiment 3

[0055] Example 3 Artificially synthesized recombinant spidroin gene MaSp2

[0056] The sequence structure of the recombinant spidroin protein selected in this embodiment is [T 1 -W n -T 2 -W n ] m , where n=1, m=4, T 1 The amino acid sequence of the spidroin domain monomer is shown in SEQ ID NO.3, T 2 The amino acid sequence of the spidroin protein domain monomer is shown in SEQ ID NO.4, the amino acid sequence of the protease cleavage site of W is ENLYFQS (the letters above are common amino acid abbreviations), and the recombinant spidroin protein The amino acid sequence is shown in SEQ ID NO.15. According to the recombinant spidroin protein sequence selected above and the principle of using preferred codons in Escherichia coli, the nucleotide encoding the recombinant spidroin protein consistent with the amino acid sequence of the recombinant spidroin protein was designed For the sequence, see the nucleotide sequence of SEQ ID NO.16 in the sequence listing.

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Abstract

The invention provides a recombinant spider silk protein. The sequence structure comprises at least two spider silk protein structural domain monomers and at least one protease enzyme digestion site;each protease enzyme digestion site is positioned between every two adjacent spider silk protein structural domain monomers. By inserting each protease enzyme digestion site between every two adjacentspider silk protein domain monomers, when the recombinant spider silk protein is used in vitro, the recombinant spider silk protein can be combined with a tool enzyme to control the enzymolysis of the recombinant spider silk protein. Compared with existing spider silk proteins which need to be slowly degraded for a long time under the assistance of microorganisms, the recombinant spider silk protein has the advantage of controllable degradation; when the recombinant spider silk protein is used in the human body, since the protease enzyme digestion sites on the recombinant spider silk proteincan be enzymatically hydrolyzed by enzymes in the human body, a biological material prepared from the recombinant spider silk protein can be slowly degraded in the human body, so that the applicationvalue of the recombinant spider silk protein is increased, and the application is wider.

Description

technical field [0001] The invention belongs to the technical field of proteins, and in particular relates to a recombinant spidroin protein and its application. Background technique [0002] Spider silk is a natural high molecular weight protein fiber with good mechanical properties and biological quality, mainly reflected in its high strength, good elasticity, light texture, high and low temperature resistance, good biocompatibility and degradability, etc. characteristics, so that it has a very wide range of application prospects. For example, research has found that spider silk is stronger than any known natural silk or artificial silk, equivalent to 5 times that of steel wire of the same volume. In terms of weight, spider silk is 25% lighter than chemically synthesized silk, but Elasticity can be extended to 10 times the original length. Due to the advantages of high strength, good elasticity and light texture, spider silk was applied to textiles as early as the 18th ce...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C09D189/00D01F4/00C08J5/18C08L89/00D21H13/34
CPCC07K14/43586C07K2319/50C08J5/18C08J2389/00C09D189/00D01F4/00D21H13/34
Inventor 李春李琳车锐
Owner SUZHOU BAIYUAN GENT CO LTD
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