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Method for establishing a system of dual-promoter methanol yeast efficiently expressing recombinant human metallothionein, and preparation and purification method of the protein

A technology of metallothionein and methanol yeast, which is applied in the establishment of a double-promoter methanol yeast high-efficiency expression system for recombinant human metallothionein and the preparation and purification of the protein, which can solve the problems of plasmid instability, lack of promoters, and poor secretion efficiency

Inactive Publication Date: 2012-09-12
汪志友
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In 1981, Hitzeman et al. successfully expressed human interferon with Saccharomyces cerevisiae, but the system has certain limitations, such as lack of a strong promoter, poor secretion efficiency, unstable plasmid, etc.

Method used

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  • Method for establishing a system of dual-promoter methanol yeast efficiently expressing recombinant human metallothionein, and preparation and purification method of the protein
  • Method for establishing a system of dual-promoter methanol yeast efficiently expressing recombinant human metallothionein, and preparation and purification method of the protein

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Experimental program
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Effect test

Embodiment 1

[0032] Embodiment two:

Embodiment 2

[0034]Select a single colony of GS115 methanolic yeast cultured on the YEPD plate with dual promoters secreting and expressing the recombinant I-type human metallothionein rhMT-1, and culture it in a shaking flask placed on a shaker at 30°C for 12-24 hours to OD600nm =0.20-0.60, then transferred to a shake flask containing 1.5L of yeast nitrogen base medium and seed medium, and fermented at 28-30°C for 24 hours. Then transfer to 30L fermenter with 5-10% inoculum size for cultivation. To the fermentor was added 13.5 L of salt medium containing per liter 27 ml H3PO4, 0.9 g CaSO4.2H2O, 18 g K2SO3, 15 g MgSO4.7H2O, 4.13 g KOH, 40 g glycerol, 4.4 ml trace mineral solution. During the fermentation process, adjust the pH value with ammonia water to maintain it at 5.0-5.5. Oxygen flow and stirring at 30°C, the stirring speed is 900-1000rpm, and an appropriate amount of defoamer is added to maintain the dissolved oxygen concentration above 30%. After 24 hours of fermentation, add gly...

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Abstract

The invention discloses a method which is feasible in pharmaceutical industry for efficient secretion expressing, preparing and purifying of gene recombinant human metallothionein or analogues thereof. The method is characterized by introducing exogenous genes containing dual-promoters into relevant host yeast cells in transformation or double crossover recombination manner by using plasmid vector pPICZ alpha-MT of exogenous metal sulfur protein genes which contain methanol response element AOX and contain or not contain metal response element MRE to construction expression engineering cells containing the dual-promoters in the methanol yeast host cells. According to the invention, several problems with traditional preparation method of metallothionein, such as high production cost, slaughter and organ harvesting to large number of animals, virus pollution derived from animal, endotoxin pollution derived from bacterial, immunogen of non-humanized protein and the like, are avoided effectively,.

Description

technical field [0001] The present invention relates to a method for establishing a dual-promoter methanolic yeast highly expressive gene recombinant human metallothionein (rhMT) system embedded with methanol response elements and metal response elements, and using the system to express, prepare and purify the target protein-metallothionein The technology of protein belongs to the field of biopharmaceuticals, and its key is to transfect a kind of carrier plasmid pPICZα-MT containing methanol response element AOX and exogenous metallothionein gene with or without metal response element MRE through calcium phosphate transfection ( Transformation), electric shock transfection or homologous recombination (double crossover recombination), etc., the exogenous gene containing the double promoter is introduced into the relevant host yeast cells, the purpose is to provide a suitable for industrial large-scale low-cost production of human metal sulfur Feasible implementation of protein....

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/66C12N1/19C12P21/02C07K14/825C07K1/14C12R1/84C12R1/78C12R1/88
Inventor 汪志友
Owner 汪志友
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