Prokaryotic cell non-fusion soluble expression system

A prokaryotic cell and expression system technology, applied in the field of prokaryotic cell non-fusion soluble expression system, can solve the problems such as low yield of inclusion body denaturation/renaturation biological activity and increased production cost.

Inactive Publication Date: 2007-08-15
SHENYANG PHARMA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] Based on the current situation of non-fusion expression of exogenous target genes in prokaryotic cells, especially the solution of inclusion bodies rich in disulfide bonds in the expression products, when E. coli cells are

Method used

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  • Prokaryotic cell non-fusion soluble expression system
  • Prokaryotic cell non-fusion soluble expression system
  • Prokaryotic cell non-fusion soluble expression system

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Experimental program
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Embodiment 1

[0028] According to the measures and principles of the above-mentioned object of the present invention, the non-fusion expression plasmid pSYPU-1 in prokaryotic cells is produced by T 7 -lac (lactose) double promoter and T 7 Transcription unit regulated by terminators. The translation of each gene in the transcription unit is regulated by prokaryotic SD sequence, translation initiation codon and translation termination codon respectively. The most upstream of the transcription unit is a multiple cloning site region with independent translation regulation, which is used for recombination (cloning) of various exogenous target genes pre-expressed, and its purpose is to enable the high-efficiency expression of exogenous target genes. Immediately following this region is a thioredoxin reductase TrxA gene with independent translational regulation. The construction measures and principles are implemented according to the model of transcription unit I above. The transcription unit ...

Embodiment 2

[0031] According to the above measures and principles of the present invention, the construction of prokaryotic cell non-fusion expression plasmid pSYPU-1a is the same as pSYPU-1 in principle and form, the difference is that the antibiotic resistance gene is ampicillin.

[0032] The exogenous target gene expressed is analgesic-antitumoral peptide I (AGAP I), AGAP I is composed of 74 amino acid residues, with a molecular weight of 7820Da, containing eight cysteine ​​residues, forming four pairs of disulfides key. The AGAP I gene is recombined in the multi-cloning site region of the prokaryotic cell non-fusion expression plasmid pSYPU-1a, the AUG start codon is added before the AGAP I gene, and the translation stop codon is added at the end of AGAP I expression. The obtained recombinant pSYPU-1a-AGAP I expression plasmid was transformed into prokaryotic cell expression host cell BL21(DE3), and the bacterial cells were collected after IPTG-induced expression (37°C culture inducti...

Embodiment 3

[0034] According to the above measures and principles of the present invention, the construction of prokaryotic cell non-fusion expression plasmid pSYPU-1b is the same as pSYPU-1 in principle and form, the difference is that the antibiotic resistance gene is chloramphenicol.

[0035] The recombinant pSYPU-1b-ANEP II expression plasmid was transformed into the E. coli expression host cell BL21(DE3), and the expression was induced by IPTG (induction at 37°C for 3 hours). The bacteria were collected and lysed by ultrasonication, and centrifuged to obtain two parts of the supernatant and the precipitate. . Cell lysates, ultrasonic supernatants, and precipitates were analyzed by small molecule Tricine-SDS-PAGE. The imaging system analysis showed that the expression product of ANEP II accounted for more than 12% of the total bacterial protein, and ANEP II was mainly present in the supernatant of ultrasonically lysed bacteria, accounting for more than 90% of the total expression.

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Abstract

The invention discloses a procaryotic cell non-merge solubility pressing system, which comprises the following steps: building one type auxiliary protein gene or two type auxiliary protein gene and pre-expressing exogenesis goal gene in a transcription unit; proceeding intracellular co-express through each self-contained translational control system of auxiliary protein gene and pre-expressing exogenesis goal gene in the same pronucleus expression host cell; utilizing the auxiliary protein to realize non-merge solubility expression especially to realize non-merge solubility expression with multiple disulfide bond peptide chain. This system can make the expressing quantity of procaryotic cell occupy above 105 of thallus total protein under the normal condition.

Description

Technical field: [0001] The present invention relates to the field of biotechnology. To be precise, it is a prokaryotic non-fusion soluble expression system Background technique: [0002] With the in-depth understanding of the molecular mechanism of gene expression in Escherichia coli in the 1970s, the high-efficiency expression of exogenous target genes in Escherichia coli became possible. At the same time, the expression system technology of Escherichia coli was also the first to be used in the production of genetic engineering drugs. Due to the clear genetic background of Escherichia coli, fast propagation, low cost, high expression level, easy operation and many other advantages, most of the exogenous target genes and genetic engineering drugs are currently produced by Escherichia coli expression system technology. Based on overcoming the deficiencies of Escherichia coli endotoxin, lack of post-translational modification, and easy folding errors and formation of inclusi...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/74
Inventor 张景海刘岩峰吴春福
Owner SHENYANG PHARMA UNIVERSITY
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