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Expression of recombined human protein disulphide isomerase (hPDI491) with Pichia pastoris in secretion manner

A disulfide bond isomerase and protein technology, applied in the direction of isomerase, recombinant DNA technology, application, etc., to achieve the effect of high expression, high activity, and reducing the formation of protein inclusion bodies

Active Publication Date: 2012-06-06
张洁
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, there is no report at home and abroad to use the method of gene cloning to produce human protein disulfide isomerase (human protein disulphide isomerase, hPDI) with the help of methanol yeast engineering bacteria 491) related stories

Method used

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  • Expression of recombined human protein disulphide isomerase (hPDI491) with Pichia pastoris in secretion manner
  • Expression of recombined human protein disulphide isomerase (hPDI491) with Pichia pastoris in secretion manner
  • Expression of recombined human protein disulphide isomerase (hPDI491) with Pichia pastoris in secretion manner

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Design and construction of recombinant plasmids, preparation of r-hPDI 491 protein

[0027] (1) Clone hPDI 491 cDNA gene, construction of expression plasmid pPICZα-hPDI 491

[0028] According to hPDI 491 Gene sequence, design primer sequence. The target gene was amplified from the human liver cDNA library by PCR method. The amplified product was excised from the restriction endonuclease site, and recombined with the expression plasmid pPICZα of Pichia pastoris to construct the expression plasmid pPICZα-hPDI 491 , Transform NovaBlue host bacteria. Extract the plasmid, and then analyze the corresponding restriction endonuclease site 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 positive clones.

[0029] The restriction endonuclease used in the present invention was purchased from Takara Company, the methanolotrophic Pichia strai...

Embodiment 2

[0049] Example 2: r-hPDI 491 Protein Bioactivity Assay

[0050] r-hPDI 491 Protein-catalyzed granule monolineage colony-stimulating factor (GM-CSF) folding assay. After the GM-CSF inclusion body protein extracted with urea was purified by SephacrylS-200, it was added to the TKM buffer at a concentration of 1 mg / mL, and then 4 mg / mL r-hPDI was added 491 and 10 5 mol / L DTT, acted at 25°C for 10 hours, measured the activity without adding r-hPDI 491 The GM-CSF activity in the above system was used as a control, and the molecular state was detected by gel exclusion HPLC. At the concentration of GM-CSF 1mg / mL with Cu 2- Oxidative refolding, the specific activity of GM-CSF was 4.2×10 6 u / mg, monomer peak area accounted for 62%, r-hPDI 491 Under protein catalysis, the specific activity can be increased to 8.5×10 6 u / mg, the monomer peak area increased to 92%.

[0051] DNA and protein molecular sequences

[0052] SEQ-1 sequence description:

[0053] (1) Sequence description...

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Abstract

The invention which belongs to the biotechnological field concretely relates to recombined hPDI491, a preparation method thereof and an application thereof. According to the invention, an RT-PCR process is applied to amplify to obtain hPDI491 cDNA, the hPDI491 cDNA is recombined with a Pichia pastoris expression vector pPICZalpha to obtain an expression plasmid pPICZalpha-hPDI, the pPICZalpha-hPDI is converted into Pichia pastoris X33, and high expression engineering strains are screened out. The expression of a large amount of the recombined hPDI491 in Pichia pastoris in secretion manner is realized. The purity of the engineering strains which are fermented, induction-expressed, and separation-purified is greater than 90%. Experiments proves that the hPDI491 has an in vitro correct folding promotion effect on other proteins.

Description

technical field [0001] The invention belongs to the field of biological technology, in particular to a recombinant human protein disulfide isomerase (human protein disulphideisomerase, hPDI 491 ) Construction of engineering strains, fermentation, purification of expression products and other preparation methods and applications. Background technique [0002] In the process of developing and producing protein drugs by genetic engineering, the problem of Inclusion Bodies (IB) is often encountered, that is, a large number of exogenous proteins expressed by engineered bacteria are densely aggregated to form a water-insoluble structure. Proteins in inclusion bodies are biologically inactive and need to be refolded to become active proteins. The formation of inclusion bodies is very important because there are many sulfur-containing amino acids in the recombinant protein, which cannot be folded correctly. However, disulfide bonds play a very important role in the stability of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/61C12N9/90C12Q1/533
Inventor 张洁
Owner 张洁
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