Spider dragline silk protein and biosynthesis method thereof

A spider silk protein and biosynthesis technology, applied in the biological material and biological field, can solve the problems such as fusion of spider silk protein and collagen which has not yet been seen, and achieve the effects of being beneficial to separation and purification, reducing production costs and facilitating cultivation.

Inactive Publication Date: 2018-03-16
JINGGANGSHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Taking advantage of the characteristic of collagen-like protein to form a triple helix structure in a directional way, fusing it with other proteins to improve the physical and chemical properties of each other has aroused the interest of some researchers, but there has been no report on the fusion of spidroin and collagen

Method used

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  • Spider dragline silk protein and biosynthesis method thereof
  • Spider dragline silk protein and biosynthesis method thereof
  • Spider dragline silk protein and biosynthesis method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Construction, expression and product purification of spidroin gene:

[0047] According to GenBank's spider silk protein gene sequences AY555585 and AH015065, the structural characteristics of the spidroin gene sequence and the degeneracy of codons, the corresponding DNA sequence was modified on the original basis, and the spidroin gene monomer S was designed. And use the Garnier method of Antheprot software to predict the secondary structure of the protein. Add recognition sites for isotail enzymes DraIII and Van91I at both ends of the gene monomer S; design adapters A1 (A1-1, A1-2) and A2 (A2-1, A2-2) a total of four single-stranded DNA fragments, A1 -1 and A1-2 contain the recognition site of EcoRI, and A2-1 and A2-2 contain the recognition site of NotI. Design (Pro-Gly-Pro) n Collagen-like protein homopolymer extension gene, the trimer repeat number n is determined to be 15, and the recognition sites of homologous enzymes DraIII and Van91I are added at b...

Embodiment 2

[0067] Example 2: Structural characterization analysis of spidroin-like proteins

[0068] Mass spectrometry analysis was performed on the isolated and purified spidroin, and the results were as follows: image 3 As shown, the results of mass spectrometry analysis show that the molecular weight of the pseudospiderin is 52032.46Da; the theoretically designed pseudospiderin contains 615 amino acids, and the molecular weight is 52242.93Da; the exact molecular weight of the actual pseudospiderin and the designed pseudospider The theoretical molecular weight of the protein is similar.

[0069] Infrared spectroscopic analysis was carried out on the isolated and purified spidroin, and the results were as follows: Figure 4 As shown, from the wave numbers of amide A, amide B, amide I, amide II, and amide III in the infrared spectrum analysis, it can be judged that the pseudo-spider silk protein has similar structural features to the natural spidroin protein.

[0070] The circular dic...

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Abstract

The invention provides a spider dragline silk protein and a biosynthesis method thereof. The spider dragline silk protein comprises an 'ABA-type' triblock copolymer of a spider silk protein and a collagen-like protein, the amino acid sequence and the nucleotide sequence of the spider dragline silk protein are SEQ ID NO.1 and SEQ ID NO.2, and the collagen-like protein is a tripolymer (Pro-Gly-Pro)15 and positioned at two ends of the spider silk protein. The biosynthesis method includes the steps: (1) designing and optimizing a spider dragline silk protein gene; (2) building a gene expression vector; (3) converting, fermenting and culturing engineering bacteria; (4) inducing expression; (5) extracting and purifying products. The spider dragline silk protein has good biocompatibility, directing properties and fibroblast potential and can meet requirements of artificial spider silk fiber molecular weight, and the spider dragline silk protein is mild in biosynthetic process and simple in process and provided with a potential protein fiber material and has a broad prospect of industrial implementation.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the field of biomaterials, in particular to a spidroin protein fused with a structure of spidroin and collagen capable of directional self-assembly and a biosynthesis method thereof. Background technique [0002] Spider silk is one of the toughest natural fibers known so far, and its main component is spidroin protein. As a natural protein fiber, spider silk has biodegradability and temperature tolerance, and has excellent mechanical properties; spider silk also has the ability to resist ultraviolet radiation and special anti-solubility, which can resist most hydrolytic proteases role. The excellent mechanical properties and mechanical properties make spider silk have great application potential in high-performance materials such as space shuttle composites, military protective clothing, and parachute ropes. At the same time, the good biocompatibility and biodegradability of spider ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70
CPCC07K14/43518C07K14/78C07K2319/00C12N15/62C12N15/70
Inventor 刘斌
Owner JINGGANGSHAN UNIVERSITY
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