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Method for improving production efficiency of insulin and its analogues

A technology of insulin glargine and its precursor, which is applied in the field of biopharmaceuticals and can solve problems such as limiting the scope of application

Active Publication Date: 2020-09-08
烟台普罗吉医药科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this medium can only be used in an environment with a pH greater than 6.8, which limits its application range

Method used

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  • Method for improving production efficiency of insulin and its analogues
  • Method for improving production efficiency of insulin and its analogues
  • Method for improving production efficiency of insulin and its analogues

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: recombinant insulin glargine fusion protein expression

[0049] Referring to Novagen's "pET System Operation Manual" (10th edition), the insulin glargine fusion protein plasmid was constructed according to conventional molecular cloning techniques, and transferred into pET30a BL-21(DE3) Escherichia coli expression strain to obtain the insulin glargine fusion protein engineering bacteria , fermented recombinantly expressed insulin glargine fusion protein, under 100L fermentation volume, OD 600 reached 150, the target protein expression rate can reach 42% ( figure 1 ).

Embodiment 2

[0050] Embodiment 2: Purification of denatured protein of insulin glargine fusion protein

[0051] The insulin glargine fusion protein fermentation liquid obtained according to the method of Example 1 was centrifuged (9000rpm x 20min), the collected bacteria were crushed three times with a high-pressure homogenizer, and the precipitate was collected by centrifugation (9000rpm x 30min). Resuspend the pellet with a resuspension buffer containing 20mM Tris, 2M urea, 5mM EDTA, 10mM dithiothreitol, and 1% Triton-100, homogenize again three times with a high-pressure homogenizer, and collect the pellet by centrifugation (13000rpm x 30min) . The obtained inclusion bodies were dissolved with 8M urea, and 20mM dithiothreitol was added.

[0052] Using hydroxyapatite medium (such as CHT TM Ceramic Hydroxyapatite) carries out chromatographic purification to the denatured protein obtained by the above-mentioned method. Refer to Bio-Rad's "CHT TMCeramic Hydroxyapatite InstructionManual...

Embodiment 3

[0053] Embodiment 3: Refolding of insulin glargine fusion protein

[0054] Dilute the insulin glargine fusion protein denatured protein solution obtained according to the method of Example 2 into the refolding buffer to obtain the insulin glargine fusion protein refolding protein, and the refolding buffer contains 20mM NaH 2 PO 4 , 20mM NaAc, 5mM GSH, 1mM GSSG, the pH value is 5.0. When the concentration of the target protein is 10mg / ml, the renaturation rate is 91%. Concentrate the obtained insulin glargine fusion protein refolding protein solution and perform buffer replacement, the replacement buffer contains 20mM NaH 2 PO 4 , 20mM Tris, pH 8.0. The results of RP-HPLC detection of renaturation rate are shown in image 3 .

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Abstract

The invention provides a method for improving the efficiency in preparation of insulin and the like. The method comprises the step of conducting chromatography on a precursor of insulin glargine by use of a hydroxyapatite medium. The invention further provides a preparation method for active insulin glargine. The preparation method comprises the step of conducting enzyme digestion on a recombinant expression precursor of insulin glargine by use of a Kex-2p enzyme. In a preferred embodiment of the invention, the preparation method comprises the following steps: obtaining the recombinant expression precursor of insulin glargine; conducting enzyme digestion on the recombinant expression precursor of insulin glargine by use of the Kex-2p enzyme; conducting further chromatographic purification on the enzyme digestion product, so as to obtain active insulin glargine.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a preparation method of long-acting insulin analogue insulin glargine. Background technique [0002] Diabetes is a major disease that threatens human health, and the global incidence is increasing year by year. Insulin is a key drug in the treatment of diabetes, which can effectively control blood sugar and has been widely used and accepted clinically. In order to meet different usage needs, people have developed a variety of insulin analogs, providing patients with a variety of choices in terms of onset time. [0003] Insulin and its analogues contain two peptide chains, A and B, connected to each other by disulfide bonds. In the process of recombinant expression, insulin and its analogs usually exist in the form of fusion proteins, that is, the two peptide chains of A and B are connected by a connecting peptide (ie, chain C). After purification and the fusion protein with co...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/62C07K1/22C12P21/06
CPCC07K14/62C12P21/06
Inventor 常国栋周代福窦鑫刘鹏
Owner 烟台普罗吉医药科技有限公司
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