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Preparation method of recombinant human insulin

A technology for recombining human insulin and proinsulin, applied in insulin, chemical instruments and methods, recombinant DNA technology, etc., can solve the problems of amino acid residues at enzyme cleavage sites, low production efficiency, poor stability, etc., and achieve simple and convenient conversion process, The effect of saving production cost and simplifying the process flow

Active Publication Date: 2020-07-28
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(2) There are a large number of proteolytic enzymes in E. coli cells, which makes the expressed target protein easy to be degraded, especially small molecular proteins such as insulin, which have poorer stability
[0009] However, the currently used fusion protein technology fails to solve the following three outstanding technical problems in insulin production: (1) cannot significantly improve the denaturation and renaturation efficiency of proinsulin protein outside the cell; Design a specific enzyme cleavage site (a short peptide with a length of about 10 to 20 amino acids and a specific sequence) between the original (target protein) so that the target can be released by proteolytic digestion (such as thrombin, enterokinase) in the subsequent process protein
However, the cleavage of these enzymes usually results in the residue of some amino acids at the cleavage site, which affects the biological function of the target protein; and the enzymes used to excise these fusion tags generally have the problems of poor specificity and low cleavage efficiency; (3) Proinsulin after denaturation and refolding treatment still needs to go through a three-step enzyme cleavage reaction (ie fusion tag cutting enzyme, trypsin and carboxypeptidase) to obtain active insulin
Therefore, the process of industrial production of insulin is still very complicated, the production efficiency is very low, and the production cost has been high

Method used

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  • Preparation method of recombinant human insulin
  • Preparation method of recombinant human insulin
  • Preparation method of recombinant human insulin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Design of Sumo-proinsulin fusion gene

[0043] Based on the pET-SUMO vector, the pET-SUMO-Proinsulin recombinant plasmid with the Sumo tag was designed and constructed ( figure 1 a), the plasmid includes T7 promoter, Lac control element, proinsulin gene and Sumo gene coding region (SEQ ID No.1).

[0044] In order to test the effect of Sumo fusion tag on the expression of proinsulin in Escherichia coli, the pET-Proinsulin (proinsulin) plasmid without Sumo tag was constructed in addition ( figure 1 b), the plasmid includes T7 promoter, Lac control element, proinsulin gene coding region (SEQ ID No.3). The specific method is as follows:

[0045] A DNA fragment (SEQ ID No.2) with a full length of 357 bases was obtained by chemical synthesis. This fragment has two endonuclease sites, EcoRI and HindIII, at the 5' and 3' ends, respectively, and the middle sequence contains 28 amino acids at the C-terminal of the compiled Sumo and the full length of human proinsul...

Embodiment 2

[0046] Induced expression of embodiment 2 recombinant Sumo-proinsulin protein

[0047] The pET-Proinsulin and pET-SUMO-Proinsulin recombinant plasmids constructed and confirmed by DNA sequencing were transferred into BL21(DE3) cells by the traditional heat shock method, single clones were selected, and grown in LB medium at 37°C to OD 600 =0.5, then add 1 mM IPTG to induce expression for 6 hours.

[0048] In order to compare the effect of the Sumo fusion tag on the expression of proinsulin protein in Escherichia coli, after the induction, the bacterial solution was centrifuged and the obtained large intestine was lysed, and then analyzed by gel electrophoresis. The results are shown in figure 2 . figure 2 Among them, lane M represents the marker; lane 1 represents Pet-Sumo (positive control), with a relative molecular mass of 17kDa; lane 2 represents Pet-Insulin (C peptide), with a theoretical relative molecular mass of 9kDa, and the arrow shows an ideal situation The pos...

Embodiment 3

[0049] Example 3 Denaturation and renaturation of recombinant Sumo-proinsulin

[0050] After the induction in Example 2, the Escherichia coli expressing the recombinant Sumo-proinsulin protein was lysed, and the precipitate was collected to obtain insoluble inclusion bodies. At the same time, polyacrylamide gel electrophoresis analysis was performed on the supernatant and the precipitate, and the results were as follows: image 3 shown. image 3 Among them, lane M represents the marker; lane 1 represents the uninduced E. coli lysate after plasmid transfer; lane 2 represents the supernatant after the first wash with 1×PBS; lane 3 represents the first wash with 1×PBS Precipitate after washing; Lane 4 indicates the supernatant after the second wash with 1×PBS; Lane 5 indicates the precipitate after the second wash with 1×PBS; Lane 6 indicates the supernatant after washing with washing solution; Lane 7 indicates the supernatant after washing with washing solution precipitation. ...

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Abstract

The invention relates to a preparation method of recombinant human insulin. The preparation method comprises: inserting a nucleotide sequence shown in the formula of SEQ ID No. 2 into a position between EcoRI and HindIII enzyme sites of a pET-SUMO vector to obtain a recombinant plasmid, transferring the recombinant plasmid into a host strain, culturing the host strain in a medium at 18 to 37 DEG Cuntil OD600 of 0.5-0.7, carrying out induced expression, separating a Sumo-proinsulin fusion protein inclusion body, washing the Sumo-proinsulin fusion protein inclusion body through a buffer solution containing urea, carrying out centrifugation, taking precipitates, carrying out denaturation and gradient renaturation to obtain a renatured Sumo-proinsulin fusion protein, and carrying out one-stepdigestion renaturation on the Sumo-proinsulin fusion protein through Sumo protease, trypsin and carboxypeptidase B at pH of 6.0-8.0 at a digestion temperature of 16-37 DEG C for digestion time of 3-6h to obtain the recombinant human insulin.

Description

technical field [0001] The invention relates to the technical field of recombinant protein production, in particular to a method for preparing recombinant human insulin. Background technique [0002] Insulin is produced by human pancreatic beta cells and is an important hormone used to regulate the balance of carbohydrate and fat metabolism in the body. As the first genetically engineered protein drug approved by the US Food and Drug Administration (FDA), recombinant human insulin has important uses and broad application prospects in the clinical treatment of diabetes. According to the World Health Organization, there are currently 420 million people with diabetes in the world, and another 160 million people are pre-diabetic. In the next 20 years, global insulin consumption will increase from the current US$12 billion to US$54 billion. [0003] Insulin is a short peptide molecule consisting of 51 amino acids with a relative molecular mass of only 5808 Daltons. However, du...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/70C07K19/00
CPCC07K14/62C07K2319/00C12N15/70
Inventor 龙乔明
Owner SUZHOU UNIV
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