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A kind of preparation method of insulin glargine and its analogues

A technology for insulin analogues and insulin glargine, which is applied in the field of preparation of insulin glargine and its analogues, can solve the problems of low yield, low separation yield, high cost, etc., achieve high yield, wide application range, and method easy effect

Active Publication Date: 2016-03-09
SHANGHAI HUAYI BIO LAB CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that the theoretical yield of the two structures formed after the precursor is transpeptidized is 50% each, and the structures are relatively consistent, and the separation yield is low.
The reaction conditions of Arg introducing protecting groups are all acidic about 12°C (refrigeration is required), and the yield is low. In its examples, the yield of Arg adding protecting groups is reported as > 60%
Moreover, US2009 / 0192073 uses basic amino acids containing protective groups, and the cost is too high

Method used

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  • A kind of preparation method of insulin glargine and its analogues
  • A kind of preparation method of insulin glargine and its analogues
  • A kind of preparation method of insulin glargine and its analogues

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Embodiment 1: Utilize Escherichia coli expression system to construct genetically engineered bacteria

[0052] The preferred codons of Escherichia coli were selected, and the gene fragment expressing the precursor of insulin glargine was obtained by fusion PCR technology. The amino acid sequence translated was FVNQHLCGSHLVEALYLVCGERGFFYTPKTRRKEAEDLQVGQVELGGGPGAGSLQPLALEGSLQRKGIVEQCCTSICSLYQLENYCG. GSK connection, the fusion sequence selected in this embodiment is a segment of staphylococcal protein A (SPA), MADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNK. After introducing the NdeIEcoRI enzyme cutting site and connecting it into the expression vector pET17b, the genetically engineered bacteria expressing the precursor of insulin glargine were obtained after conventional genetic engineering. figure 2 , with figure 2 The target protein band in the expression is the fusion protein of the expressed insulin glargine precursor, and the fusion protein of the...

Embodiment 2

[0053] Example 2: Construction of genetically engineered bacteria using Pichia pastoris expression system

[0054] The preferred codons of Pichia pastoris were selected, and the gene fragment expressing the precursor of insulin glargine was obtained by fusion PCR technology, and the amino acid sequence translated was FVNQHLCGSHLVEALYLVCGERGFFYTPKTRRKGIVEQCCTSICSLYQLENYCG. In order to improve the cleavage efficiency of the signal peptide and ensure the integrity of the N-terminus, the Add the connection fragment KREEAEAEAEPK, introduce the XhoIEcoRI restriction site and connect it into the vector pPIC9, then clone it into the expression vector pPIC9k with SalI / SacI, and obtain the genetically engineered bacteria expressing the precursor of insulin glargine after conventional genetic engineering. Take the fermentation broth for TricineSDS-PAGE analysis, the results are shown in the attached image 3 , with image 3 The target protein band in is the secreted and expressed insuli...

Embodiment 3

[0055] Embodiment 3: crude pure expression product

[0056] Escherichia coli expression product in Example 1, with 50mMPB+2mMEDTA, pH7.5 breaks up the bacteria; With 50mMTris-HCl+2mMEDTA+0.5%TritonX-100, pH8.0; 50mMTris-HCl+2mMEDTA+1MUrea, wash successively with pH8.0 package; denatured with 50mMTris-HCl+2mMEDTA+8MUrea, pH9.0; sulfonated with 50mMTris-HCl+2mMEDTA+8MUrea+0.3MNa2SO3, pH9.0; renatured with 50mMGly+2mMEDTA+1MUrea+1mMβ-Me, pH9.5, Put on the Q column, and obtain the crude and pure precursor protein after pH 8.0 salt concentration gradient elution, SDS-PAGE analysis, the results are shown in the appendix Figure 4 , the results show that the precursor protein with higher purity can be obtained after renaturation crude purification, the molecular weight is about 17.8KD, and the main impurity is the dimer formed in the renaturation process.

[0057] In Example 2, the yeast expression product was diluted and adjusted to pH 4.0, and eluted with pH and salt gradient on a...

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Abstract

The invention discloses a preparation method of insulin glargine and an analogue thereof. The preparation method includes the following steps: (1) a gene engineering method is used for preparing a precursor of the insulin glargine and the analogue of the insulin glargine with a chain B and an end C containing a plurality of basic amino acids; (2) an amino acid side chain protective agent is used for distinguishing arginine or lysine through pancreatic enzyme specificity, and the insulin glargine and the analogue of the insulin glargine are provided with protecting groups and obtained under the effect of the protective agent and pancreatic enzyme; or the specificity is used for acting on clostripain of the arginine (Arg) or endoproteinase lysine (Lys) C of the Lys directly without protection; (3) carboxypeptidase is added optionally to remove unprotected basic amino acids at the tail end of the C; and (4) the glargine and the analogue of the insulin glargine are obtained through deprotection. The preparation method is simple and convenient, high in yield, wide in application range and suitable for introduction of more than two basic amino acids.

Description

technical field [0001] The present invention relates to a preparation method of insulin glargine and its analogues, in particular, the present invention relates to a method for preparing recombinant insulin glargine and its analogue precursors by genetic engineering, and converting the precursors into A method for insulin glargine and its analogs, the main structural feature of which is that the C-terminus contains multiple basic amino acids. Background technique [0002] Insulin glargine replaces asparagine (Asn) at position 21 on human insulin A chain with glycine (Gly); adds 2 arginines to threonine (Thr) at position 30 in B chain (Arg) and synthetic human insulin analogues. The addition of arginine increases the positive charge on the surface of the insulin molecule, and the isoelectric point rises from 5.4 to 6.7, making insulin glargine appear as a colorless and transparent solution in a weak acid environment, while its solubility is nearly neutral under physiological...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/81C12P21/06C07K14/62
CPCY02P20/55
Inventor 夏晶
Owner SHANGHAI HUAYI BIO LAB CO LTD
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