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Method for purifying recombinant proteins with intein-mediated elastin like proteins

An elastin-like and recombinant protein technology, applied in the field of recombinant drug protein purification, can solve problems such as difficulty, cost increase, limited clinical application, etc., and achieve the effect of simple operation and high trial cost.

Inactive Publication Date: 2012-03-14
EAST CHINA UNIV OF SCI & TECH
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Problems solved by technology

Second, resins and separation equipment are expensive, which increases the overall cost, making large-scale recovery and purification of proteins still expensive and difficult
However, the clinical application of the mouse monoclonal antibody is severely limited because it easily induces the human anti-mouse antibody (HAMA) immune response in the human body.

Method used

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  • Method for purifying recombinant proteins with intein-mediated elastin like proteins
  • Method for purifying recombinant proteins with intein-mediated elastin like proteins
  • Method for purifying recombinant proteins with intein-mediated elastin like proteins

Examples

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Embodiment 1

[0030] Example 1: Intein-mediated elastin-like purification system Purification of recombinant protein TAT-Survivin (T34A) and TAT-Anti-VEGF single-chain antibody (ScFv).

[0031] 1. PCR amplification of the TAT-Survivin(T34A) / TAT-Anti-VEGF single-chain antibody gene.

[0032] According to the sequences of plasmids pRest-TAT-Survivin (T34A), PET28a-Anti-VEGF single-chain antibody and PET-ELP-intein (given to Dr. Wood from Duke University, USA) constructed in our laboratory, the following primers were designed For the amplification of the above genes. Its sequence is shown in Table 1 below. The primers used were synthesized by BGI Corporation.

[0033] Table 1 Primers used in this experiment

[0034]

[0035] The experiment technique of molecular biology was used, and the specific method was carried out in "Molecular Cloning". Build according to the following technical routes and methods. figure 1 Construct a schematic road map for the plasmid, and finally construct a g...

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Abstract

The invention discloses a method for purifying recombinant proteins with intein-mediated elastin like proteins (ELPs). In the method, a method which does not need any affinity matrix or label excision enzyme is developed by combining the temperature-dependent phase transfer characteristics of the ELPs and the label self-excision function of intein. The method is easy to operate, and has low cost and extremely good application prospect.

Description

technical field [0001] The present invention relates to a method for purifying recombinant drug protein, more specifically, relates to a method for purifying recombinant protein drug by using intein-mediated elastin-like protein through non-chromatographic method. Background technique [0002] Since the 1990s, gene recombination technology has been greatly developed, especially in recent years, with the need for protein research and production, gene recombination technology has advanced by leaps and bounds, and many genetic engineering products have appeared, which have profoundly affected our human beings and their environment. As the downstream engineering of gene technology, the separation and purification technology of gene recombinant protein has shown its importance more and more. The cost of separation and purification of genetic engineering products accounts for about 60% to 80% of the total cost, so the research on new technologies and strategies for efficient prote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C07K19/00C07K16/22C07K1/14C12R1/19
Inventor 马兴元唐莉莉石磊夏鸿西
Owner EAST CHINA UNIV OF SCI & TECH
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