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Human proinsulin fusion protein and preparation method of human insulin

A technology of human insulin and fusion protein, applied in the field of genetic engineering, to achieve high yield, promote renaturation, and less by-products

Inactive Publication Date: 2012-07-11
SHANGHAI KEXIN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] Technical problems to be solved by the present invention Problems such as Arg-A0 insulin by-products and renaturation existing in the prior art

Method used

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  • Human proinsulin fusion protein and preparation method of human insulin
  • Human proinsulin fusion protein and preparation method of human insulin
  • Human proinsulin fusion protein and preparation method of human insulin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Embodiment 1, preparation of human insulin

[0093] This example describes a preparation and purification of human proinsulin and the conversion of human proinsulin to

[0094] Method for maturing human insulin. The steps involved in this process are described in detail below.

[0095] a) Fermentation: Transform the expression vector of cloned human proinsulin into Escherichia coli BL21(DE3), and culture the transformed Escherichia coli in shake flasks and high-density fermentation cultures. In both cases, up to 20% of the total cell protein can be observed -30% expression.

[0096] b) Harvest inclusion bodies: human proinsulin is expressed in the form of inclusion bodies in Escherichia coli. Inclusion bodies are protein-based aggregates insoluble in water, in which a large number of highly expressed foreign proteins are enriched. The protein is human proinsulin in this example. The harvested cells were resuspended in Tris salt buffer, homogenized twice using a high...

Embodiment 2

[0104] Example 2. Human proinsulin expression vector construction and C peptide mutation

[0105] The present invention introduces a human proinsulin expression system based on the T7 system, and the T7 vector used here is pCRT7NT (available for purchase from Invitrogen).

[0106] According to the amino acid sequence of human insulin, the following piece of DNA (SEQ ID NO7) was chemically synthesized, which contained the 5' end NdeI and the 3' end EcoRI restriction site, and encoded human proinsulin with a histidine tag:

[0107] catatgcaccaccatcatcaccacggtggccgctttgttaatcagcacctgtgcggctctcacctggtagaggcgctgtatctggtttgtggcgaacgtggcttcttctacactaaaccgacccgtcgcgaagcggaggacctgcaagtgggccaggtcgaactgggtggcggtccgggtgctggttccctgcagccgctggccctggaaggttctctgcagaaacgtggtatcgtggaacagtgctgcacgtctatttgtagcctgtaccagctggaaaactactgcaactaagaattc

[0108] This DNA was subcloned into the pCRT7NT plasmid, placing human proinsulin under the control of the T7 promoter and SD sequence. The expression s...

Embodiment 3

[0129] Example 3 Preparation of insulin lispro analogs

[0130] This example describes a method for preparing and purifying proinsulin lispro and converting proinsulin lispro into mature insulin lispro.

[0131] Insulin lispro differs from human insulin in that Pro28 and Lys29 are interchanged. Since the Pro28 position is a key site for the formation of human insulin multimers, amino acid substitutions in insulin lispro make the analogues mostly exist in the form of monomers. Since insulin can bind to insulin receptors and regulate blood sugar only in the form of monomers, insulin lispro, which mainly exists in the form of monomers, has a faster onset of action than ordinary insulin, and is a fast-acting insulin.

[0132] The proinsulin sequence of insulin lispro with novel C peptide used in the present invention is as follows:

[0133] MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTKPT RREAED LQVGQVELGGGPGAGSLQPLALEGSLQSR GIVEQCCTSICSLYQLENYCN

[0134] In addition, due to the su...

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Abstract

The invention belongs to the technical field of the gene engineering, and particularly relates to a human proinsulin fusion protein and a preparation method of human insulin. The human proinsulin fusion protein comprises X-B-C-A four peptide chains from an N terminal to a C terminal, wherein the X peptide chain is deleted or a guide peptide, the B-C-A peptide chains have amino acid sequences shown by SEQ ID NO.3 or 90% homologous with amino acid sequences shown by SEQ ID NO.3 and mature body of the human proinsulin fusion protein comprises mutant with hypoglycemic activity, and is characterized in that: the Lys 64 residues on the C terminal of the C peptide are substituted by non basic amino acids. In the human proinsulin fusion protein, Arg-A0 insulin by-products formation is avoided, wherein the C peptide can promote the proinsulin renaturation. The preparation method has the following advantages: relatively less required steps, relatively less by-products and relatively high yield of the insulin.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a human proinsulin fusion protein and a preparation method of human insulin. Background technique [0002] Insulin is a non-glycosylated heterologous polypeptide dimer linked by disulfide bonds. It consists of 51 amino acids, including 21 amino acid A peptide and 30 amino acid B peptide. It consists of A7-B7 and Two pairs of disulfide bonds between A20-B19 are connected, and there is also an intra-chain disulfide bond of A6-A11 in the A peptide. ( figure 1 ) [0003] Insulin is synthesized by pancreatic β-cells in the form of proinsulin, and the C-terminus of the B peptide in proinsulin is connected to a single chain by another peptide chain, C-peptide and A-peptide N. The C-peptide contains 35 amino acids in total, and its N-terminal is Arg-Arg (RR) and its C-terminal is Lys-Arg (KR). Mature insulin is cleaved by enzymes secreted by β cells. Three enz...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/21C12N1/19C12N5/10C07K14/62C12R1/19
Inventor 傅海龙李忠秀孙祥明包骏
Owner SHANGHAI KEXIN BIOTECH
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