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Recombinant spider silk protein and preparation method and application thereof

A spider silk protein and protein technology, applied in the field of recombinant spider silk protein, can solve the problems of lower gene expression efficiency, lower expression level, large molecular weight of spider silk protein, etc.

Active Publication Date: 2018-08-28
GENSUN INST OF BIOMEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the molecular weight of natural spider silk is very large (>300KDa), and when the foreign protein expressed by E. coli is greater than 60KDa, the expression level will decrease. The molecular weight of natural spider silk protein is too large, and it is almost impossible to express successfully.
For example: Fahnestock et al. reported the expression of spider silk protein in Escherichia coli, but found that the expression efficiency of genes larger than 3kb was reduced, and there was a phenomenon of gene deletion expression

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

[0068] Example 1 Construction of recombinant spider silk protein Escherichia coli expression strain

[0069] This patent involves the construction of multiple recombinant spider silk protein strains. The splicing of repeat modules (repeat modules refer to REPA, ADF3Rep) is realized by the molecular operation of homologous enzymes. The scheme flow is as follows figure 1 shown. The hybrid concatemer (ADF3Rep1-REPA4) formed by ADF3Rep and four REPA concatemers is used as an example to illustrate, as follows:

[0070]1. Referring to the ADF3 mRNA sequence published in GenBank (GenBank gene accession number: U47855.1), intercept the core repeat region and entrust GenScript Biotechnology Co., Ltd. to target the gene sequence Codon optimization was carried out in the E. coli expression system to obtain the nucleotide sequence of a single ADF3Rep, as shown in SEQ ID NO: 5. In order to facilitate subsequent molecular operations, restriction sites BamH I were introduced at the 5' and ...

Embodiment 2

[0081] Example 2 High-density fermentation of recombinant spider silk protein Escherichia coli strain

[0082] Spider silk protein contains a large number of repetitive amino acid sequences. This characteristic leads to problems such as tRNA pool depletion, gene replication barriers, and protein translation truncation when recombinantly expressed in E. coli, resulting in low expression levels. Studies have shown that as the molecular weight of artificial spidroin protein increases (greater than 60KDa), the difficulty of protein expression will increase greatly, and the problem of expression failure may occur. The molecular weight of natural spidroin protein can reach more than 200KDa. Although there is no direct research to show that the molecular weight of spidroin protein is proportional to its mechanical properties, it is still necessary to make the molecular weight of artificially designed spidroin protein as close as possible to that of natural spidroin protein. similar. ...

Embodiment 3

[0089] Example 3 Purification and Preparation of Recombinant Spider Silk Protein

[0090] This part describes the non-chromatographic protein purification method of spider silk protein. After purification, the purity of recombinant spider silk protein (electrophoretic purity) can reach about 70%. There is no obvious effect on other operations. Using the fermented thalline prepared in Example 2 to operate through the same purification process can achieve a similar purification effect, and the specific purification operation is as follows:

[0091] 1. Take a certain amount of high-density fermented bacteria and resuspend them to 100g / L with buffer A (20mM Tris 100mM sodium chloride 1mM EDTA1.5M urea, pH8.0), and use a high-pressure homogenizer to sterilize the above-mentioned bacteria suspension. broken body.

[0092] 2. Centrifuge the above cell lysate at 10,000 g for 30 min in a low-temperature centrifuge, discard the supernatant, and collect spider silk protein inclusion bo...

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Abstract

The invention discloses recombinant spider silk protein, a coding gene thereof, an expression and purification method and application of the recombinant spider silk protein in the fields of silk formation, film formation and the like. The recombinant spider silk protein consists of an N-terminal non-repeated area (N-NRT), a core repeated area (REP), and a C-terminal non-repeated area (C-NRT), wherein the core repeated area consists of 1-3 ADF3Reps and / or 1-6 REPAs. A series of novel spider silk protein sequences designed by using the recombinant spider silk protein integrate the main characteristics, such as high strength and high elasticity, of spider silk. Moreover, the sequences are introduced into escherichia coli through a genetic engineering technology separately, a corresponding high-density fermentation process is established, the high-efficiency prokaryotic expression of the spider silk protein is achieved, and the fermentation yield can reach 0.5 g / L or above. In addition, aninventor develops a non-chromatographic protein purification method according to the properties of the spider silk protein, and the purity of the purified recombinant spider silk protein meets the requirements of silk formation or film formation.

Description

technical field [0001] The present invention relates to a recombinant spider silk protein, its encoding gene, expression and purification methods, and the application of the recombinant spider silk protein in silk formation, film formation and the like. Background technique [0002] Spider silk is both hard like steel and elastic like rubber. Its outstanding properties are mainly manifested in: high strength, high elasticity, high fracture work, it can be said to be the strongest material so far, known as "biological steel". With the in-depth research on spider silk, it is found that spider silk also has the characteristics of biodegradability, super shrinkage, high temperature resistance, low temperature resistance and compatibility with biological tissues. Due to the unique physical and biological properties of spider silk, it has broad application prospects in medicine, materials, military and textiles. The artificial spider silk produced by Swedish scientist Jan Johans...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K1/30C12N15/70C12N1/21C12P21/02D01D5/00C12R1/19
CPCC12N15/70C12P21/02D01D5/0015C07K14/43518Y02A50/30
Inventor 马永赵百学王安良
Owner GENSUN INST OF BIOMEDICINE
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