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Method for reinforcing secretion efficiency of recombination exogenous protein in sprout fungi expression system

An exogenous protein and high-efficiency technology, applied in the direction of recombinant DNA technology, biochemical equipment and methods, enzymes, etc., can solve the problems of increased cost of recombinant exogenous protein, prolonged culture period, lower culture temperature, etc., and achieve the goal of secretion efficiency Effect

Inactive Publication Date: 2009-07-01
MOGAM BIOTECH RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, such methods of lowering the culture temperature have disadvantages associated with increased production costs of recombinant foreign proteins due to prolonged culture periods

Method used

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  • Method for reinforcing secretion efficiency of recombination exogenous protein in sprout fungi expression system
  • Method for reinforcing secretion efficiency of recombination exogenous protein in sprout fungi expression system
  • Method for reinforcing secretion efficiency of recombination exogenous protein in sprout fungi expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Comparison of intracellular galactose transport capacity between yeast hosts A and B

[0063] Yeast host A (Saccharomyces cerevisiae 2805, genotype: MATα pep4::HIS3 prb1-Δ1.6Rhis3-Δ200 ura3-52 GAL2 can1, Sohn, J.H. et al. Proc. Biochem., 30:653-660, 1995; and Kim , T.H.et al.Biotechnol.Lett.24:279-286, 2002) and yeast host B (Saccharomyces cerevisiae BJ3501, genotype: MATα pep4::HIS3 prb1-Δ1.6R his3-Δ200 ura3-52gal2 can1, ATCC 208280, USA) were streaked on a YPD agar plate and placed in an incubator at 30° C. for about 18 hours, and colonies were isolated. Each colony of yeast hosts A and B was inoculated into separate YPG [yeast extract 1% (w / v), peptone 2% (w / v) and galactose 2% (w / v)] liquid medium and Shake culture in an incubator at 30°C for about 80 hours. To determine the consumption of galactose over time, the residual galactose concentration in the medium was determined by quantifying reducing sugars via 3,5-dinitrosalicylic acid (DNS) analysis (Mo...

Embodiment 2

[0065] Example 2: Construction of Yeast Transformants Secreting Foreign Proteins Using Yeast Hosts A and B

[0066] 2-1. Using Yeast Host A, Construction of Yeast Transformant Saccharomyces cerevisiae 2805 / MδLK8 Secreting Foreign Protein

[0067] Using the expression vector pMBRI-LK8 (Korean Patent Publication Laid-Open Publication No. 2004-0069840) used by the present inventors to produce recombinant LK8 in Pichia pastoris, co-synthesis of the α-factor secretion signal and LK8 cDNA was performed. separate. This application is hereby incorporated by reference in its entirety. The pMBRI-LK8 vector was treated with EcoR I for 7 hours and washed using a PCR purification kit (Qiagen, USA). Then, the vector was treated with BamHI for 7 hours, and the DNA was separated by gel electrophoresis. Using a gel extraction kit (Qiagen, USA), a DNA fragment having the α-factor secretion signal of SEQ ID NO: 4 and the LK8 cDNA sequence of SEQ ID NO: 16 was obtained. The DNA fragment thus ...

Embodiment 3

[0072] Example 3: Comparison of exogenous protein secretion capacity of transformed yeast strains A and B in fed-batch culture using galactose as carbon source

[0073] In the case of galactose fed-batch culture of the yeast transformants A and B in Example 2, the following analysis was performed to compare the secretion efficiency of the foreign protein. First, the transformed yeast strain as the master seed was treated with YPD [yeast extract 1% (w / v) and peptone 2% ( w / v)] culture medium for 24 hours, so that the desired cell mass and activity (20-fold dilution, OD 600 = 0.8 to 1.2). After seed culture in YPD medium, the seed culture solution was inoculated into the starter medium. First, the batch culture stage is a cell growth stage and an adaptation stage to galactose used as an expression inducer of foreign protein LK8. For this purpose, the cells were given a galactose acclimatization period while the cells were allowed to proliferate by inoculating more than 1% (v / ...

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Abstract

Provided is a method for improving secretion efficiency of a recombinant foreign protein in a yeast expression system. The method comprises transforming a yeast host with a recombinant foreign gene construct comprising a galactose-inducible promoter, a secretion signal sequence and a gene encoding the foreign protein to construct a transformed yeast strain; and culturing the transformed yeast strain under the condition that the activity of the galactose-inducible promoter is controlled. Improved secretion efficiency of the foreign protein can be achieved by decreasing over-expression-induced insoluble precipitation of the recombinant foreign protein suffered by a conventional galactose-inducible promoter-based yeast expression system, via appropriate control of a level of galactose functioning as an inducer of the galactose-inducible promoter in cells. Due to improved secretion efficiency of the recombinant foreign protein, present invention makes a contribution to improvement in productivity of recombinant foreign proteins in the yeast expression system and reduction in production costs.

Description

technical field [0001] The invention relates to a method for improving the secretion efficiency of recombinant foreign protein in a yeast expression system. Background technique [0002] The use of microorganisms to produce large amounts of foreign proteins is the most important technical field in the protein pharmaceutical industry. Recombinant foreign proteins can be expressed intracellularly, or alternatively can be secreted extracellularly, in order to recover and purify the desired product. When expressing proteins intracellularly, overexpression of proteins in many cases can often lead to intracellular accumulation of proteins in an inactive and water-insoluble form, and can bring various disadvantages of yield-reducing factors, such as rupture of solid microorganisms. Cumbersome procedures and complex and difficult purification procedures to isolate the desired protein from various host proteins present in the cell. However, extracellular secretion of the desired pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/04C12N9/38
CPCC12N15/81
Inventor 林滢权金圣根
Owner MOGAM BIOTECH RES INST
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