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Spider dragline silk protein optimized expression method

A technology of traction silk protein and spider silk protein, applied in the field of expression and production optimization of traction silk protein MaSp, which can solve the problem of low expression

Inactive Publication Date: 2015-09-30
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invention can effectively overcome the defect of low expression level of highly repetitive recombinant spider silk protein in the production and fermentation process; it provides a good reference for the research and application of fermentation production of spider silk protein and similar highly repetitive sequence proteins

Method used

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  • Spider dragline silk protein optimized expression method
  • Spider dragline silk protein optimized expression method
  • Spider dragline silk protein optimized expression method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] In this example, two recombinant spider silk protein expression vectors, recombinant MaSp1 and recombinant MaSp2, were respectively constructed, in which the strains, plasmids, enzymes and culture medium used were as follows: the expression plasmids were pET19b and pET-28a(+), and the expression host was large intestine Bacillus Escherichia.coli BL21 (DE3), the cloning host is Escherichia coli DH5; genetic manipulation tools include: restriction endonuclease, DNA polymerase, T4 DNA ligase; LB medium.

[0046] The component contents of the LB medium are: 10g / L tryptone, 5g / L yeast powder, and 10g / L sodium chloride.

[0047] like figure 1 As shown, the method of constructing the recombinant plasmid in this example is as follows: the strategy of "end-to-end splicing" is adopted, that is, two homologous enzymes NheI and SpeI are used for seamless splicing, and a spider with a molecular weight of 28.3kDa to 256.5kDa is synthesized. Drag silk proteins, including MaSp2-8-mer,...

Embodiment 2

[0049] This example uses different temperatures to express and produce recombinant spider traction silk protein MaSp2-64 polymer in a shake flask system, wherein the strain is Escherichia coli BL21(DE3) / pET28a-MaSp2-64 polymer obtained in Example 1

[0050] This embodiment includes the following steps:

[0051] 1) Take out the frozen strain from -80°C, that is, Escherichia coli BL21(DE3) / pET28a-MaSp2-64 polymer, and inoculate it into 2 mL of LB medium (containing antibiotic concentration: 50 mg / mL of kanamycin), Cultivate for about 14 hours at 30°C and 220rpm.

[0052] 2) Inoculate 1 mL of seed liquid into a 250 mL shake flask containing 100 mL of LB medium or R / 2 medium at a ratio of 1:100, and culture at 30°C and 220 rpm until the initial logarithmic growth (OD 600 =0.4‐0.5);

[0053] The component content of the R / 2 medium: 2g / L diammonium hydrogen phosphate, 6.75g / L potassium dihydrogen phosphate, 0.85g / L citric acid, 5mL / L trace metal solution, 10g / L glucose, 0.7 g / L m...

Embodiment 3

[0060] The strains used in this example are Escherichia coli BL21(DE3) / pET28a-MaSp2-80-mer and BL21(DE3) / pET19b-MaSp1-96-mer, and the seed culture and protein production fermentation operations are the same as in Example 2, wherein MaSpI96 The antibiotic concentration is 100mg / mL; Sample processing and detection operation are identical with embodiment 2, and result is as follows:

[0061] like image 3 as shown, image 3 ‐A is the protein expression gel map of MaSp2‐80mer, image 3 ‐B is the protein expression gel map of MaSp1‐96mer. After adding the IPTG initial expression factor to the recombinant engineered bacteria that were expressed and produced at 30°C, there was almost no MaSp2-80 and MaSp1-96 protein expression; while the comparison temperature culture temperature was lower than 25°C (in this embodiment, 16 ℃ and 25℃), the yields of high molecular weight recombinant spider dragline protein MaSp2-80-mer and MaSp1-96-mer can be clearly seen, and there is a significan...

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Abstract

The invention relates to a spider dragline silk protein optimized expression method, belonging to the field of bioengineering. The method comprises the following steps: constructing high-molecular-weight recombinant spider dragline silk protein grains in Escherichia coli, and carrying out expression production, wherein the protein yield is enhanced by lowering the culture temperature in the protein expression production process; and by using the combined strategy of high-density fermentation and culture temperature lowering on the fermentation tank level, implementing the double enhancement of the cell quantity and expression level. The method can effectively overcome the defects of low expression level of the highly-repetitive recombinant spider dragline silk protein in the production fermentation process, and provides favorable reference meanings for fermentation production study and application of spider silk proteins and similar highly-repetitive sequence proteins.

Description

technical field [0001] The invention relates to a method for optimizing the expression of repetitive sequence proteins in the biological field, in particular to the optimization of expression and production of MaSp, a dragline protein of Nephilaclavipes. Background technique [0002] The natural spider pulling silk protein is a protein polymer secreted by the giant jugular gland glands in the spider, known as the "spider lifeline"; it has the mechanical properties of high strength, high elasticity and high breaking work, and has good mechanical properties. It is one of the natural biological materials with the best comprehensive performance in nature. It is mainly composed of two proteins, spider pulling silk protein 1 and spider pulling silk protein 2 (MaSp1&2). Both dragline proteins are high-molecular-weight proteins with a molecular weight distribution between 250-350 kDa; previous studies have shown that MaSp1 is mainly responsible for rigid properties and MaSp2 is res...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02
Inventor 夏小霞杨雁祥钱志刚钟建江
Owner SHANGHAI JIAO TONG UNIV
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