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Purification method and application of ELP (elastin-like polypeptides)-III type collagen

A collagen and purification method technology, which is applied in the fields of peptide preparation methods, chemical instruments and methods, microorganism-based methods, etc., can solve the problems of high cost and complex protein purification, and achieve the effect of simple method and high purification yield.

Pending Publication Date: 2021-03-16
点斗基因科技(南京)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are still many problems, such as: protein purification is complicated, it needs to be purified by a series of chromatography methods such as ion exchange and molecular exclusion, and the cost is high

Method used

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  • Purification method and application of ELP (elastin-like polypeptides)-III type collagen
  • Purification method and application of ELP (elastin-like polypeptides)-III type collagen
  • Purification method and application of ELP (elastin-like polypeptides)-III type collagen

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Embodiment Construction

[0029] The present invention is described in further detail now in conjunction with accompanying drawing. These drawings are all simplified schematic diagrams, which only illustrate the basic structure of the present invention in a schematic manner, so they only show the configurations related to the present invention.

[0030] figure 1 , figure 2 , image 3 , Figure 4 , Figure 5 and Figure 6 The purification method and application of a kind of improved ELP-III type collagen shown, the specific steps of the present invention are as follows:

[0031] 1. Construction of ELP100-CLP III fusion protein expression vector

[0032] (1) Synthesize the ELP100 gene and insert it between the BamHI and NdeI restriction sites of the pET28a vector to form the pET28a-BamH-ELP100-NdeI vector, and introduce it into DH5a Escherichia coli.

[0033] (2) Extract the pET28a vector and amplify the BamHI-ELP100-NdeI fragment by PCR, connect it to the vector pRELPN to form the pRELPN-BamHI-E...

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Abstract

The invention discloses a purification and renaturation method of fusion protein of ELPs (elastin-like polypeptides) and type III collagen (colagen III). ELP tag ELP100 and collagen III polypeptide genes are connected to a prokaryotic expression vector pRELPN to construct a pRELPN-CLP III-ELP100 fusion protein expression vector, and the vector is highly expressed in BL21 (DE3) escherichia coli. The protein exists in the escherichia coli in the form of an intracellular inclusion body, so that the fusion protein is purified and renatured by an inclusion body purification and renaturation method.The method comprises the following steps: collecting and crushing a strain; washing off impure protein, lipid and nucleic acid; finely purifying protein; performing renaturating by a titration dilution method; performing ITC (inverse transition cycling) purifying; and identifying the activity by an MTT cell method. The method is high in purification yield, simple and suitable for large-scale production.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a purification method and application of ELP-III collagen. Background technique [0002] Elastin-like polypeptides (ELPs) are genetically engineered polypeptides that have been artificially modified, have elasticity and undergo phase transitions with temperature changes, and form polymers because of their structure in series with pentapeptide repeating sequence units, namely ( Val-Pro-Gly-Xaa-Gly, VPGXG), wherein, Xaa can be any amino acid except proline, usually valine (Val, V), alanine (Ala, A), glycine (Gly, G) Leucine (Leu, L) Isoleucine (Ile, I), Lysine (Lys, K), Phenylalanine (Phe, F), Histidine (His, H) and other amino acid types, the repeating unit n usually ranges from 20-120. At present, there are two main synthesis methods, one is chemical synthesis to synthesize polypeptides, and the other is gene recombination synthesis. Compared with the chemical synthesis method, th...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70C07K1/36C07K1/34C07K1/30C12N5/077A61K8/65A61Q19/08C12R1/19
CPCC07K14/78C07K7/06C12N15/70C12N5/0656A61K8/65A61Q19/08C07K2319/50C07K2319/00C12N2533/54
Inventor 张杰
Owner 点斗基因科技(南京)有限公司