Recombinant human insulin and purification and preparation method thereof

A recombinant human insulin, ubiquitin-like technology, applied in the field of biomedicine, can solve the problems of high production cost, low renaturation rate of inclusion body expression, complicated production process, etc., to reduce production cost, realize soluble expression, and solve complex problems. low sex effect

Inactive Publication Date: 2020-12-29
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In summary, in the preparation process of recombinant human insulin, the ubiquitous problems are complex production process, low renaturation rate of inclusion body expression, and high production cost.

Method used

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  • Recombinant human insulin and purification and preparation method thereof
  • Recombinant human insulin and purification and preparation method thereof
  • Recombinant human insulin and purification and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Construction of recombinant human proinsulin gene expression vector

[0027] The recombinant human proinsulin gene sequence having the gene sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 was synthesized by a DNA synthesis company (Invitrogen).

[0028] Ligate the synthetic recombinant human proinsulin target fragment with the prokaryotic expression vector pET30a(+) (the vector has been connected with the SUMO gene), and the ligation reaction system (10 μl) is as follows:

[0029]

[0030] A total of 10 μl of the system was mixed, and connected overnight at 4°C. After enzyme digestion and identification, the recombinant human proinsulin gene expression vector pET-30a-SUMO-Insulin was constructed.

Embodiment 2

[0032] Induced Expression of Recombinant Human Proinsulin Fusion Protein

[0033] The constructed recombinant human proinsulin gene expression vector pET-30a-SUMO-Insulin was transformed into the expression strain Rosetta (DE3) (Beijing Quanshijin Biotechnology Co., Ltd., catalog number: CD801). Transformed single colonies were inoculated into 20 mL of LB medium containing ampicillin (Amp) (100ug / mL), cultured at 37°C for 8 hours, and then inoculated into another 20 mL of LB containing Amp (100ug / mL) at a ratio of 1:100. culture medium at 37°C, when the A600 is around 0.35, add isopropylthiogalactopyranoside (IPTG) to a final concentration of 0.25mmol / L for induction, the induction temperature is 20°C, harvest the cells after 4h, and use lysisbuffer (20mM Na 3 PO 4 , 20mM imidazole, 500mM NaCl, pH 7.5) to resuspend the bacterium, centrifuge after breaking the bacterium, take the supernatant and precipitate respectively for 12% SDS-PAGE electrophoresis analysis. The results ...

Embodiment 3

[0035] Purification and separation of insulin analogues

[0036] According to the conditions for the induced expression of the recombinant human proinsulin fusion protein in Example 2, expand the culture, collect the bacteria after induction and expression, suspend with lysis buffer, sonicate and then centrifuge or filter to obtain the supernatant of the bacteria.

[0037] The above supernatant was purified by affinity chromatography, Ni Sepharose 6FF was used as the chromatography medium, and the equilibrium liquid was lysis buffer. The eluent is 20mM Na 3 PO 4 , 250mM imidazole, 500mM NaCl, pH 7.5, to obtain a crude sample of SUMO-Insulin fusion protein.

[0038] Add trypsin to the crude sample of SUMO-Insulin fusion protein obtained above and mix for enzymatic digestion reaction, so that the mass ratio of trypsin and crude SUMO-Insulin fusion protein is 1:1000, react the mixture at 37°C for 1 h, and use Phosphoric acid was used to adjust the pH of the mixture to approxim...

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Abstract

The invention relates to a method for purifying and preparing recombinant human insulin, in particular to a method for purifying and preparing soluble expression recombinant human insulin. The methodcomprises the following steps: firstly, designing and synthesizing a full-length sequence of recombinant human proinsulin, connecting the full-length sequence to an expression vector containing SUMO molecular chaperone peptides, introducing the constructed expression vector into a proper host cell, performing culturing, performing inducing, expressing recombinant human proinsulin fusion proteins in a soluble form, and performing purifying through affinity chromatography, enzyme digestion, ion exchange, reversed phase chromatography and other steps to finally obtain the recombinant human insulin with the purity of 98% or above. Compared with an existing method for preparing human insulin, the method has the advantages of being easy to operate, high in yield, low in production cost and the like, and is suitable for large-scale industrial production.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to a method for preparing recombinant human insulin, in particular to a preparation method for soluble expression and purification of recombinant human insulin. Background technique [0002] Diabetes is a major disease that threatens human health worldwide. The number of diabetic patients worldwide is increasing year by year and is expected to reach 552 million in 2030. There are many types of diabetes drugs, among which insulin is still widely used as a specific drug for treating diabetes, so the consumption of insulin will also increase year by year. The clinical dosage of insulin is large, and each diabetic patient needs to use about 1.5-2.0 mg of insulin per day, and it needs to be used for life. Therefore, it has good commercial value and social significance to develop a process method for large-scale and low-cost preparation of insulin. [0003] Currently, two systems are used for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K14/62C07K1/22C07K1/18C07K1/16C12P21/06C12N15/62C12N1/21C12R1/19
CPCC07K14/62C07K2319/95C12N15/70C12P21/06
Inventor 梁鑫淼叶贤龙郭志谋于伟
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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