Variant of Kex2 enzyme and stable expression method

A stable and variant technology, applied in the biological field, can solve the problems of cumbersome protein expression induction process, low expression level, unfavorable industrial production, etc., and achieve the effect of retaining biological activity, high enzyme activity, and convenient purification

Inactive Publication Date: 2020-04-10
VONSUN PHARMATECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In previous studies, it was found that most of kex2 uses methanol-inducible yeast expression system, the protein expression induction process is cumbersome to operate, and the expression level is relatively low, which is not conducive to industrial production

Method used

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  • Variant of Kex2 enzyme and stable expression method
  • Variant of Kex2 enzyme and stable expression method
  • Variant of Kex2 enzyme and stable expression method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Embodiment 1. Kex2 protease truncation body or variant design

[0078] Kex2 consists of 814 amino acid residues (SEQ ID NO.1), the whole molecule contains signal peptide (1-19aa), precursor peptide (20-113aa residues), catalytic domain (114-410aa residues), P Domain (411-624aa residues), Ser / Thr rich domain (625-679aa residues), transmembrane region (680-699aa residues) and extracellular domain (700-814aa residues), kex2 structure Such as figure 1 shown.

[0079] The 1-613th truncated body of Kex2, based on the amino acid sequence of the full-length kex2 enzyme, wherein the 225th, 436th, and 503rd positions are mutated from K to A or P, and at the same time, the C-terminal of the truncated body is added or not Add the His tag.

[0080] The 1-667th truncated body of Kex2, based on the amino acid sequence of the full-length kex2 enzyme, wherein the 225th, 436th, and 503rd positions are mutated from K to A or P, and at the same time, the C-terminus of the truncated body...

Embodiment 2

[0087] Example 2. Construction of recombinant Pichia pastoris

[0088] The recombinant expression plasmid containing kex2 truncation or variant is linearized by Avr ll or BspH I enzyme, and then the linearized recombinant expression plasmid is recovered by using TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0.

[0089]The constructed linearized recombinant expression plasmid is transferred into Pichia pastoris X33 or gs115 competent cells by electroporation, so as to obtain recombinant Pichia pastoris.

[0090] The pGAPZa A expression plasmid contains the Zeocin antibiotic selection gene, and the recombinant Pichia pastoris that stably and efficiently secretes and expresses kex2 can be screened by using different concentrations of Zeocin antibiotics.

[0091] A YPD plate (1% Yeast Extraction, 2% Peptone, 2% glucose) with a Zeocin concentration of 100 μg / ml was prepared for screening recombinant Pichia pastoris with stable and high-efficiency secretory expression.

Embodiment 3

[0092] Example 3. High-efficiency secretory expression of Kex2 protease

[0093] From the YPD plate containing Zeocin antibiotics, randomly pick single clones that grow well, inoculate them into 3ml of YPD medium, set the culture conditions at 28-30°C, 250-300rpm, cultivate for about 24h, and then transfer to 0.2% volume Connect to fresh YPD medium, continue to cultivate, and take samples at about 24h, 48h, and 72h of culture to identify protein expression results, such as Figure 2-4 As shown (A: indicates the protein expression results in host X33, 1-18 in the figure respectively indicate the protein expression results of 1-18 clones picked; B: indicates the protein expression results in host gs115, in the figure 1-18 18 represent the protein expression results of 1-18 selected clones, respectively).

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Abstract

The invention discloses a variant of Kex2 enzyme and a stable expression method, the variant is formed by properly truncating a Kex2 amino acid sequence and performing point mutation, the variant canstably and efficiently secrete and express a target protein through a constitutive pichia pastoris expression system, and good biological enzyme activity is maintained. The fermentation process is simple, efficient, low in cost, high in product activity and suitable for commercial production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Kex2 enzyme variant and a stable expression method. [0002] technical background [0003] Kex2 enzyme is a dialkali endopeptidase, the gene comes from Saccharomyces cerevisia, belongs to the subtilisin family, and is a calcium ion-dependent serine proteolytic enzyme. Kex2 can specifically recognize the double basic amino acid residue pair Lys-Arg or Arg-Arg in a protein or polypeptide, and cut the peptide bond from the carboxyl terminal of the second amino acid residue (ie Arg-) to play a role. [0004] In yeast, the kex2 molecule is translated on the ribosome, undergoes cleavage of the signal peptide and precursor peptide, and complex O-linked and N-linked glycosylation processes to become an active protease molecule. As a precursor processing enzyme in eukaryotes, Kex2 can process and mature many mammalian hormone precursors in vivo and in vitro. Very important. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/60C12N15/57C12N15/81C12N1/19C12R1/84
CPCC12N9/60C12N15/815C12Y304/21106
Inventor 辛中帅陈慧梅文良柱王玉刚朱亮
Owner VONSUN PHARMATECH CO LTD
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