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High-performance spider-silk-like protein material and its biosynthesis method

A technology of spider silk proteins and species, applied in biochemical equipment and methods, botanical equipment and methods, filament/thread forming, etc., can solve poor mechanical properties, low expression of spider silk proteins, premature translation termination errors, etc. problem, to achieve the effect of increasing stability, increasing elasticity and hardness, and increasing the number of disulfide bonds

Active Publication Date: 2014-06-04
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the problems of low expression level and poor mechanical properties of spider silk protein produced by Escherichia coli, utilizes the characteristic that the low ploidy of spider silk protein is not suitable for producing translation premature termination error, and expresses it, and improves the elasticity of spider silk protein through site-directed mutation And toughness and other properties, obtained the mutant spidroin protein with better performance than the natural spidroin protein

Method used

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  • High-performance spider-silk-like protein material and its biosynthesis method
  • High-performance spider-silk-like protein material and its biosynthesis method
  • High-performance spider-silk-like protein material and its biosynthesis method

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1: Sequence synthesis of spidroin drag silk gene 11R26 and ZF4 and construction of recombinant vector

[0039] SEQ ID NO: 2 and SEQ ID NO: 4 were synthesized by Shanghai Sangon Co., Ltd., and EcoRI and HindIII restriction sites were introduced at the 5' and 3' ends, respectively.

[0040] The specific process of constructing the recombinant expression vector of spidroin 11R26 (wild type, control) and ZF4 (mutant) is as follows: After double-cutting the recombinant vector pMD18-T-11R26 / ZF4EcoRI / HindIII synthesized by Shanghai Sangong Co., Ltd., gel recovery reagent Kit (purchased from Omega) to recover small fragments, while EcoRI / HindIII double cut expression vector pET-30a(+) (Novagen), column purification kit (purchased from Omega) directly through the column to recover linear pET-30a(+). The previously recovered small fragments were respectively connected with the linear expression vector pET-30a(+) with T4DNA Ligase (TaKaRa) to obtain the recombinant express...

Embodiment 2

[0041] Example 2: Expression and purification of recombinant plasmids pet30a-11R26 and pet30a-ZF4 in Escherichia coli

[0042] Pick the positive clones carrying the recombinant plasmids pet30a-11R26 and pet30a-ZF4 and inoculate them to add 50 μg·mL -1 In the LB medium of kanamycin, under the condition of 180-220rpm, cultivate overnight at 37°C, transfer the engineered Escherichia coli cultured overnight to 50mL containing 50μg·mL -1 In the LB medium of kanamycin, shake culture at 37°C and 180-220rpm until OD 600 About 0.6-0.8, add the inducer IPTG to the final concentration of 0.5mmol·L -1 , continue to cultivate for 3-5 hours, and collect the bacteria by centrifugation;

[0043] Thale ddH 2 After O washing, it was dissolved with lysis buffer (10mM imidazole, 6M urea, 10mM Tris-HCl, 100mM sodium dihydrogen phosphate, pH 8.0), 130W ultrasonic disrupted bacterial cells (working time 5s, intermittent time 5s, 50min in total), 4°C , centrifuged at 10000rpm for 30min, and colle...

Embodiment 3

[0044] Example 3: Recombinant spidroin protein 11R26 and ZF4 electrospinning and mechanical property testing

[0045] Pour the purified spidroin 11R26 and ZF4 eluate into a dialysis bag, dialyze in double distilled water for 24 hours, change it every 2-3 hours, collect the precipitate, and freeze-dry it at -80°C. Dissolve the freeze-dried two kinds of spidroin proteins in hexafluoroisopropanol (100mg / ml), dissolve them with a heating mantle at 50°C for more than 12 hours, inject them into a 1ml syringe, conduct electrospinning at 23-30kV, and fix the mica sheet on an inclined 60° placed on the aluminum foil paper under the syringe, and the angle was changed so that the spun silk fell on the mica sheet, and observed by scanning electron microscope as image 3 A and image 3 as shown in B;

[0046] The atomic force microscope (Dimension3100 atomic force microscope) HARMONIX mode detects the mechanical properties of the silk on the mica sheet, compares the mechanical properties...

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Abstract

The invention belongs to molecular biology and biomaterial fields, spider dragline silk gene is used as a base for artificial directed mutation of a part of amino acid sequence, a recombinant spider silk protein is obtained by an escherichia coli overexpression method and a Ni column purification technology, and after electrostatic spinning, a spider-silk-like protein is obtained, and the spider-silk-like protein has better consistency and elasticity than a natural spider silk protein. The spider-silk-like protein is an n-polyploid such as a tri-polyploid, hexa-polyploid or other integer-polyploid of an amino acid sequence shown in SEQ IDNO:5, wherein n is an integer equal to or greater than 1.

Description

technical field [0001] The present invention belongs to the field of biomaterials. Specifically, the present invention relates to a high-performance spidroin-like protein, which is an n-ploid of the amino acid sequence shown in SEQ ID NO: 5, wherein n is equal to 1 or Integers greater than 1, the present invention also relates to methods for synthesizing high-performance protein materials using biological methods. technical background [0002] Spider silk is a natural protein fiber with mechanical properties such as high strength, high elasticity, and high fracture energy, as well as biological properties such as significant degradability and tissue compatibility. It is used in biomedicine, materials, textiles, and military equipment. All have great potential application value. As a protein fiber, spider silk has become a research hotspot for new materials since the 1990s. Spider silk has excellent mechanical properties and thermal properties. Compared with Kevlar produced...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/70D01D5/00
CPCC07K14/43518C12N15/70D01F4/00
Inventor 咸漠张海波刘炜周凤丽
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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