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Method for obtaining soluble proinsulin by utilizing Escherichia coli expression system

An expression system and Escherichia coli technology, applied in the directions of insulin, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of complicated operation and high cost, and achieve the effect of reducing production cost and improving expression and yield.

Inactive Publication Date: 2015-03-04
BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Considering the above method from the perspective of industrial application, the operation is relatively cumbersome and the cost is relatively high

Method used

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  • Method for obtaining soluble proinsulin by utilizing Escherichia coli expression system
  • Method for obtaining soluble proinsulin by utilizing Escherichia coli expression system
  • Method for obtaining soluble proinsulin by utilizing Escherichia coli expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment １

[0051] Construction of TrxA-PI fusion expression vector

[0052] (1) According to the published human-derived preproinsulin gene sequence (Genbank CAA23828.1) in the NCBI database, the codon was optimized as E. coliPreferring codons, the full gene sequence of PI was synthesized at BGI:

[0053] 1 ATG GCA CTG TGG ATG CGT CTG CTG CCG CTG CTG GCC CTG CTG GCC 45

[0054] 1 Met Ala Leu Trp Met Arg Leu Leu Pro Leu Leu Ala Leu Leu Ala 15

[0055] 46 CTG TGG GGT CCG GAT CCA GCC GCA GCA TTC GTT AAT CAG CAT CTG 90

[0056] 16 Leu Trp Gly Pro Asp Pro Ala Ala Ala Phe Val Asn Gln His Leu 30

[0057] 91 TGT GGC AGC CAT CTG GTG GAA GCG CTG TAT CTG GTT TGC GGT GAA 135

[0058] 31 Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu 45

[0059] 136 CGT GGC TTT TTC TAT ACC CCG AAA ACC CGT CGT GAA GCG GAA GAT 180

[0060] 46 Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp 60

[0061] 181 CTG CAG GTG GGC CAG GTG GAA CTG GGT GGC GGT CCA GGC GCA GGC 225

[0062] 61 Leu...

Embodiment 2

[0076] Transform different expression strains and compare the expression of fusion proteins

[0077] The plasmids verified by sequencing were transferred into three expression strains: E. coli BL21 (DE3) (Novagen), E. coli transB (DE3) (Beijing Quanshijin Company) and Escherichia coli NKYBYP. Single clones on the plate were picked and cultured overnight in LB medium. E. coli Add Amp at a final concentration of 100 μg / ml to BL21 (DE3) medium; E. coli Amp, 15 μg / ml Kan, and 12.5 μg / ml Tet were added to transB (DE3) medium at a final concentration of 100 μg / ml; Amp, 100 μg / ml final concentration was added to Escherichia coli NKYBYP medium 15 μg / ml Kan, 12.5 μg / ml Tet, 34 μg / ml Chl (the same below).

[0078] The cultured bacterial solution was inoculated into 30 ml culture medium according to the inoculum amount of 1%, and expanded at 37°C and 200 rpm. When OD 600 When = 0.8, a final concentration of 1 mM IPTG was added, induced at 37°C and 160 rpm for 8 h, and the...

Embodiment 3

[0083] Optimization of induction conditions

[0084] (1) Induction temperature

[0085] Since the TrxA-PI fusion protein has the highest expression level in Escherichia coli NKYBYP, only the induction conditions of this strain were optimized to increase the expression level of the soluble fusion protein.

[0086] The cultured bacterial solution was inoculated into 30 ml culture medium according to the inoculum amount of 1%, and expanded at 37°C and 200 rpm. When OD 600 = 0.8, the final concentration of 1 mM IPTG was added, and the bacterial solution was induced at 15°C, 20°C, 25°C, 30°C, 35°C, 160 rpm for 8 h.

[0087] Collect the cells by centrifugation at 11000 rpm for 2 minutes, wash the cells twice with triple-distilled water, add 5 ml of 1×binding buffer (0.5 M NaCl, 20 mM Tris-HCl, 5 mM imidazole, pH 8.0) to dissolve the cells, and sonicate , Sonication conditions: power 350 watts, ultrasonic for 5 seconds, interval of 10 seconds, ultrasonic 30 times until the bacte...

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Abstract

The invention discloses a method for obtaining soluble proinsulin by utilizing an Escherichia coli expression system. Through improvements on the various aspects such as construction of a TrxA-PI fusion expression vector, selection of an Escherichia coli expression strain, optimization of the induction condition, and extraction and purification of a recombinant protein, the method has the advantages that the expression of the soluble proinsulin can be effectively improved and the production cost is reduced.

Description

technical field [0001] The invention relates to the technical field of preparation of soluble proinsulin, in particular to a method for obtaining soluble proinsulin by using an Escherichia coli expression system. Background technique [0002] Diabetes mellitus (DM) is a metabolic disorder characterized by chronic hyperglycemia caused by various etiologies. It is a syndrome caused by absolute or relative lack of insulin in the body or insensitivity of target tissues to insulin. The result of long-term interaction of factors. According to the forecast of the World Health Organization (WHO), the number of diabetic patients in the world may exceed 400 million after 2020. Diabetes has become a major international and domestic medical problem due to its complex etiology, many complications, and low cure rate. It has also brought serious economic and social problems. It is called an immortal cancer by the WHO. There are many kinds of diabetes treatment drugs. Insulin has an irrep...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/17C07K14/62
Inventor 毕玉平边斐张晓玮彭振英郑玲陈高于金慧
Owner BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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