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Proinsulin containing protecting lysine and preparation method for insulin by utilizing proinsulin

A technology of proinsulin and insulin lispro, which is applied in the field of insulin and its preparation, can solve problems such as difficult separation, difficult control of reaction conditions, and adverse effects on insulin quality, so as to avoid by-products, pure production costs, simplify enzymatic hydrolysis and follow-up The effect of the purifying operation

Inactive Publication Date: 2012-06-20
SUZHOU YUANJI BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this route, an insulin by-product (DesB30-insulin) is produced that eliminates threonine at position 30 of the B chain, which has an adverse effect on the quality of insulin
In order to reduce the production of DesB30-insulin, the amount of trypsin, pH value and reaction time must be strictly controlled, making the reaction conditions difficult to control
Despite many efforts, DesB30-insulin cannot be completely avoided because there is only one threonine difference between DesB30-insulin and insulin, and it is very difficult to separate them
Now, large-scale high-performance liquid chromatography is commonly used to separate insulin and DesB30-insulin, which greatly increases the production cost of recombinant human insulin and produces a large amount of industrial waste

Method used

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  • Proinsulin containing protecting lysine and preparation method for insulin by utilizing proinsulin
  • Proinsulin containing protecting lysine and preparation method for insulin by utilizing proinsulin
  • Proinsulin containing protecting lysine and preparation method for insulin by utilizing proinsulin

Examples

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preparation example Construction

[0055] In the method for preparing proinsulin with protected lysine at position 29 of the present invention or insulin with protected lysine at position B29, for translation in a translation system, the translation system can be Any known translation system that can be used to translate proinsulin or insulin may be an in vitro translation system, or may be a prokaryotic or eukaryotic expression system. Those skilled in the art can understand that such a system includes a suitable buffer system and enzymes related to protein synthesis. For example, Escherichia coli or yeast expression systems commonly used in the art can be used in the method of the present invention. For example, expression systems that can be used to express proinsulin or insulin of the present invention include, but are not limited to, Escherichia coli, Bacillus subtilis, baker's yeast, Pichia pastoris, insect cells and animal cells.

[0056] In a specific embodiment, the human proinsulin gene sequence with...

Embodiment 1

[0073] 1. Experimental materials:

[0074] The biological carrier-Escherichia coli mutant strain BL21 (DE3) used to express proinsulin and the deoxynucleic acid polymerase Platinum Pfx used for DNA amplification were purchased from Invitrogen;

[0075] The endodeoxynuclease-NdeI and SacI used for plasmid construction, calf intestinal alkaline phosphatase-CIP, deoxynucleic acid ligase-T4 DNA ligase, deoxynucleic acid molecular ruler, and protein molecular ruler were purchased from NEB Biolabs;

[0076] LB medium and Nε-(tert-butoxycarbonyl)-lysine were purchased from Sigma;

[0077] Kits for plasmid mini-extraction were purchased from Qiagen.

[0078] Other reagents not identified were from VWR.

[0079] 2. Experimental method:

[0080] 1. Plasmid construction

[0081] Two plasmids for expressing human proinsulin with Nε-(tert-butoxycarbonyl)-lysine at position 29 of proinsulin in Escherichia coli variant BL21(DE3) are as follows: figure 2 and image 3 As shown (the cons...

Embodiment 2

[0101] In this example, the steps of Example 1 were repeated with the same operation, except that the amber codon UAG in Example 1 was replaced with ocher codon UAA; UUA Instead of tRNA in Example 1 CUA ; Replace Nε-(tert-butoxycarbonyl)-lysine in Example 1 with Nε-(benzyloxycarbonyl)-lysine; replace SEQ ID NO.5 with SEQ ID NO.9; replace SEQ ID NO.5 with SEQ ID NO.11 replaces SEQ ID NO.4; And the steps of removing Nε-(benzyloxycarbyl)-lysine from insulin are as follows: 0.1 M bromic acid (purchased from VWR) was added to the acid insulin solution, and ammonium bicarbonate was added after 2 hours to terminate the reaction.

[0102] The results of this example were similar to those of Example 1. According to SDS-PAGE analysis and subsequent mass spectrometric analysis, the purity of the finally produced insulin reached 97%. The expression level of proinsulin measured by BCA protein concentration analysis of mature human insulin was 48 mg / L.

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Abstract

The invention relates to proinsulin with protecting amino acid at the 29 position, insulin with protecting amino acid at the B29 position and genes of the proinsulin and the insulin. The invention further relates to a preparation method for the proinsulin with the protecting amino acid at the 29 position, a preparation method for the insulin with the protecting amino acid at the B29 position, application of the proinsulin with the protecting amino acid at the 29 position and application of the insulin with the protecting amino acid at the B29 position.

Description

technical field [0001] The present invention relates to the field of recombinant insulin. More specifically, the present invention relates to insulin with protected amino acids, its preparation method and use. Background technique [0002] Insulin, especially recombinant insulin such as human recombinant insulin, has a wide range of applications in the field of medicine. Currently, there are two routes for the production of recombinant human insulin - the "chain combination" route and the "proinsulin" route. In the “chain combination” route, the two peptide chains that make up human insulin—chain A and chain B—are synthesized through biological recombination, and then chain A and chain B are mixed to generate disulfide bonds to generate biologically active human insulin. However, in this route, the efficiency of producing biologically active human insulin by directly mixing two peptide chains is relatively low. The "proinsulin" route is now more commonly used. In this rou...

Claims

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Application Information

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IPC IPC(8): C07K14/62C12N15/17C12P21/02
Inventor 万为刘文设
Owner SUZHOU YUANJI BIOLOGICAL TECH
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