Preparation method and spinning of high-concentration recombinant spider silk protein spinning solution

A technology of spidroin and solution, which is applied in the field of artificial spider silk preparation, which can solve the problems of long-term, unsuitable high-molecular-weight recombined spidroin, environmental pollution, etc., and achieves improved toughness, difficulty in aggregating into disordered precipitates, and stable properties Effect

Active Publication Date: 2020-07-03
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The centrifugal concentration method takes a long time and easily leads to disordered aggregation of high molecular weight spidroin proteins during the concentration process; dissolving spidroin proteins with strong solvents such as hexafluoroisopropanol will cause environmental pollution and lead to high molecular weight spidroin proteins. The degradation of the spidroin protein and the loss of the function of the terminal domain, so the above method is not suitable for the preparation of the spinning solution of the natural high molecular weight recombinant spidroin protein with two terminal domains

Method used

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  • Preparation method and spinning of high-concentration recombinant spider silk protein spinning solution
  • Preparation method and spinning of high-concentration recombinant spider silk protein spinning solution
  • Preparation method and spinning of high-concentration recombinant spider silk protein spinning solution

Examples

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Embodiment 1

[0037] Such as figure 1 As shown, the recombinant spidroin protein used to prepare artificial spider silk involved in this embodiment is NTD-(PR) 16 -Molecular architecture of the CTD.

[0038] This embodiment relates to the preparation method of the above-mentioned recombinant spidroin solution, comprising the following steps:

[0039] Step 1) Escherichia coli cells expressing recombinant spidroin protein were resuspended in a wall-breaking buffer solution containing 2 mol thiourea and 8 mol urea at a mass ratio of 1:10, stirred for 12 hours, and then centrifuged to collect the supernatant to obtain the recombinant Spider silk protein mixture A;

[0040] Step 2) Adjust the pH of the mixed solution A obtained in step 1 to 4.0 to precipitate some impurities, and collect the supernatant by centrifugation to obtain the recombinant spidroin protein mixed solution B;

[0041] Step 3) Dialyzing the mixed solution B into a buffer solution containing 0.3 mol of sodium chloride to o...

Embodiment 2

[0052] This example relates to high component NTD-(PR) 64 -The preparation method of CTD recombinant spidroin protein solution, comprises the following steps:

[0053] Step 1) Escherichia coli cells expressing recombinant spidroin protein were resuspended in a wall-breaking buffer solution containing 2 mol thiourea and 8 mol urea at a mass ratio of 1:10, stirred for 12 hours, and then centrifuged to collect the supernatant to obtain the recombinant Spider silk protein mixture A;

[0054] Step 2) Adjust the pH of the mixed solution A obtained in step 1 to 4.0 to precipitate some impurities, and collect the supernatant by centrifugation to obtain the recombinant spidroin protein mixed solution B;

[0055] Step 3) Dialyzing the mixed solution B into a buffer solution containing 0.3 mol of sodium chloride to obtain the reconstituted spidroin protein mixed solution C;

[0056] Step 4) Heating the mixed solution C in a water bath at 80° C. for 20 minutes to precipitate some impuri...

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Abstract

The invention discloses a preparation method and spinning of a high-concentration recombinant spider silk protein spinning solution. The molecular structure of a recombinant spider silk protein is NTD-(RP)n-CTD, an integer n represents the number of repetitions of an RP repetitive structural domain, and the amino acid sequence of the RP repetitive structural domain is shown as Seq ID No.1 or Seq ID No.2. The method can prepare the high-concentration recombinant spider silk protein spinning solution efficiently at low cost, and obtain artificial spider silks which are easy to modify and excellent in mechanical property.

Description

technical field [0001] The invention relates to a technology in the field of biopolymers, in particular to a method for purifying recombinant spidroin protein with a concentration of 50-500 mg / mL, separating and concentrating liquid-liquid phases, and a method for preparing artificial spider silk. Background technique [0002] As one of the strongest biological materials, spider silk has superior toughness than most synthetic materials due to its high strength and good ductility. Nephila clavipes traction silk is composed of two high-molecular-weight spidroin proteins, MaSp1 and MaSp2, of which more than 80% are MaSp1. Preparation of high-concentration protein spinning solution is one of the important prerequisites for biomimetic spinning of recombinant spidroin protein. [0003] In the prior art, the purified low-concentration spidroin solution is concentrated to a high concentration by centrifugation and biomimetic spinning is performed; or the purified spidroin solution ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K1/36C07K1/34C07K1/30D01F4/00
CPCC07K14/43518D01F4/00
Inventor 夏小霞胡春飞钱志刚
Owner SHANGHAI JIAO TONG UNIV
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