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Novel process for genetic engineering preparation of insulin and insulin analogs

A technology for insulin and insulin precursors, applied in the field of bioengineering, can solve the problems of low expression of gene recombination, mismatch of disulfide bonds, and high cost of chemical synthesis

Inactive Publication Date: 2008-10-29
费俭
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical synthesis method is costly, while the genetic recombination method has disadvantages such as low expression, disulfide bond mismatch, and complicated preparation of the final product.

Method used

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  • Novel process for genetic engineering preparation of insulin and insulin analogs
  • Novel process for genetic engineering preparation of insulin and insulin analogs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Construction of expression vector

[0078] The codon usage of P. pastoris is basically similar to that of S. cerevisiae, but there are some differences in individual amino acids such as Glu [Zhao, X., Huo, K.K. and Li, Y.Y.(2000). Synonymous codon usage in Pichia pastoris Sheng Wu Gong Cheng XueBao16(3):308-11.]. Chemical synthesis of monomeric insulin B following the codon preference of P. pastoris 27 K-DTrI precursor gene fragment ( Figure 1A ).

[0079] Chemically synthesize code B by the same method 1 A B 27 K-DTrI( Figure 1B ) and (DesB 1 )B 27 K-DTrI DNA ( Figure 1C ). Similar to S.cerevisiae, a suitable prepro-leader is helpful for the secretion and expression of insulin precursors in P.pastoris, and the chemically synthesized DNA fragments were cloned into the EcoR I and Not I sites of the pPIC9K plasmid (Invitrogen Company) to form α-Mating factor Ieader-EAEAYVEFK-MIP expression frame. Among them, the EA-rich spacer peptide (such as EAEAYVEFK, SEQ...

Embodiment 2

[0082] Plasmid transformation and screening of MIP expression strains

[0083] 3 μg pPIC9K / B linearized with Bg1II 27 K-DtrI plasmid electrotransformed Pichia pastoris GS115 (his4) (purchased from NRRL, deposit number NRRL Y-15851, US6,730,499), 0.4cm electric shock cup, 2.4KV, 5.3ms, two consecutive electric shocks, transformed yeast cells Painted MD board. Plasmid pPIC9K / B 27 After K-DtrI is linearized by Bgl II, after entering yeast cells, part of it will be re-circularized and integrated into the chromosome through single-point insertion to obtain Mut+ phenotype transformants, and the other part of the linear plasmid will be integrated into the chromosome through substitution to obtain the Muts expression. Type transformants, during which multi-copy insertion integration will spontaneously form. Pick 1500 single clones from the MD plate, culture them in 2ml of YPD medium at 30°C for 24 hours, take 2μL and put them on the YPD plates containing 0.5, 1, 2, and 4 mg / ml of G...

Embodiment 3

[0086] 2L scale fermentation

[0087] High-density fermentation is an effective means to obtain a large amount of heterologous protein from methanol yeast. Methanol yeast can grow in a simple inorganic salt medium. Harmful products such as alcohol and acetic acid are rarely produced during the fermentation process, so it is suitable for large-scale high-density fermentation . The pH value of the fermentation broth has a significant impact on the growth state of the cells and the stability of the secreted protein. The pH range of the fermentation was preliminarily optimized by shaking table experiments to be 4.5-6.5, and the optimum pH value was 5 ( image 3 ).

[0088] Each liter of basic inorganic salt medium (basal salt medium, BSM) contains: 50ml glycerol, 26.7mlH 3 PO 4 , 0.93g CaSO 4 2H 2 O, 18.2 g K 2 SO 4 , 14.9 g MgSO 4 ·7H 2 O), and 4.13 g KOH, autoclaved with NH 4 OH to adjust the pH value.

[0089] The formula of trace element solution (PTM1) is 1L contai...

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Abstract

The invention provides a novel process for genetic engineering preparation of insulin and insulin analogs, which comprises enzyme cutting insulin precursor with parenzyme, then isolating insulin, wherein the insulin precursor comprises the following elements from amino end to carboxyl end, (1) spacing peptide element with formula (Xaa)mZ, (2) insulin B chain element, (3) joint peptide element with formula (Xaa)nZ, and (4) insulin A chain element.

Description

technical field [0001] The present invention relates to the field of bioengineering. More specifically, the present invention relates to the genetic engineering preparation method of novel insulin and insulin analogues. Background technique [0002] Insulin has been used to treat diabetes since the 1920s, and it is still an irreplaceable diabetes drug. There are now more than 120 million people with diabetes in the world, and it is expected to be 300 million by 2025, of which 10% are type I diabetes. Each patient with type I diabetes needs 1.4-2.1 mg of insulin per day, and patients with type II diabetes also have a considerable demand for insulin. Developed countries consume 4,600 kilograms of insulin per year. In addition, some new drug delivery methods can improve the patient's medication compliance, but the bioavailability is low, and more insulin is required compared with injection. Therefore, while preventing and controlling diabetes, it is imperative to develop a m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C07K14/62C12N15/17C12N15/63
Inventor 张友尚费俭丁金国崔大敷蔡国强石嘉豪
Owner 费俭
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