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Method for performing fusion expression on protein containing disulfide bond

A fusion expression and protein technology, applied in the field of bioengineering, can solve problems such as inability to fold natural structures

Active Publication Date: 2012-07-04
SHANGHAI KAIYANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is speculated that the recombinant proteins expressed in the Pichia pastoris system cannot be folded into their natural structures in the absence of the corresponding source of folding enzymes, which leads to the above-mentioned problems

Method used

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  • Method for performing fusion expression on protein containing disulfide bond
  • Method for performing fusion expression on protein containing disulfide bond
  • Method for performing fusion expression on protein containing disulfide bond

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Design and construction of recombinant plasmids and expression of fusion proteins

[0058] (1) Clone hPDI 508 cDNA gene

[0059] Referring to the hPDI gene sequence, primers were designed, and the full-length hPDI gene was amplified from a commercial human liver cDNA library by PCR. The amplified product was cut out from the restriction endonuclease Xho I and NotI sites, and recombined with the expression plasmid pPICZα of Pichia pastoris to construct the expression plasmid pPICZα-hPDI 508 , Transform NovaBlue host bacteria. Extract the plasmid, and then analyze the corresponding restriction endonuclease sites to screen the plasmid with characteristic fragments, and then use nucleotide sequence analysis to confirm the correct position and sequence of gene recombination, and obtain the hPDI full-length gene. Positive clone.

[0060] The restriction endonuclease used in the present invention comes from Takara Company, the commercialized human liver cDNA lib...

Embodiment 2

[0070] Embodiment 2: the purification of fusion protein

[0071] 1. CM Sepharose Fast Flow column chromatography

[0072] Equilibrate the column with 50mM HAc-NaAc (pH 4.2), dilute the sample solution containing the target protein until the conductance is consistent with the equilibrium buffer, adjust the pH to 4.2, start loading the sample, and wash 6 columns with the equilibrium buffer after loading volume, and then eluted with 50mM HAc-NaAc (pH 4.0) + 1M NaCl, collecting 2 column volumes.

[0073] 2.Chelating Sepharose Fast Flow Chromatography

[0074] Dilute the CM eluent to 1 time with water, then adjust the pH to 7.4 with sodium hydroxide, put it on the Chelating Sepharose Fast Flow column that has been equilibrated with 50mM Tris-HCl (pH 7.4)+0.5M NaCl, and use the equilibration buffer after loading Rinse 6 column volumes, then wash away impurities with 50mM Tris-HCl (pH 7.4) + 20mM imidazole, and finally elute with 50mM Tris-HCl (pH 7.4) + 200mM imidazole, and collec...

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Abstract

The invention relates to a method for performing fusion expression on a protein containing a disulfide bond, in particular to a method for performing fusion expression on a recombinant protein, in particular a eukaryotic recombinant protein of which the activity is closely related with the forming of the (intrachain and interchain) disulfide bond in an eukaryotic host cell. An exogenous recombinant protein is expressed in a pichia pastoris expression system by a method for fusing human protein disulphide isomerase (hPDI) or a mutant of the hPDI. The method is suitable for expressing a eukaryotic exogenous protein of which protein activity or protein folding (comprising forms of a monomer, a dimmer or a polymer) are resistant to the disulphide bond. The protein expressed by the mode can be secreted out of a cell easily, and is high in yield, products cannot be gathered on a large scale and can be captured and purified easily, and the obtained target protein is uniform in N tail end, and high in activity. The recombinant protein expressed by the method can be industrially produced on a large scale.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a method and application for correctly expressing recombinant proteins in eukaryotic host cells, in particular to correctly expressing recombinant proteins whose biological activity is closely related to the formation of disulfide bonds (intra-chain and inter-chain). Nuclear recombinant protein. Background technique [0002] As early as the 1970s and 1980s, it was reported that a post-translational enzyme (Post-Translational Enzymes)---Prolyl 4-hydroxylase, namely prolyl-4-hydroxylase, P4H for short. It can repeat the characteristic sequence of collagen family protein (Gly-X-Y) n Y in is modified by hydroxylation (when Y is a proline residue) to become hydroxyproline. Only when a certain number of proline residues are modified into 4-hydroxyproline, the three polypeptide chains of collagen can be correctly folded into a triple helical domain, that is to say, o...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N15/81C07K19/00
Inventor 张洁王建权余彩霞
Owner SHANGHAI KAIYANG BIOTECH
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