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Method for quickly screening yeast with high-expression target proteins

A high-expression, purpose-based technology, applied in the direction of microbial-based methods, botany equipment and methods, biochemical equipment and methods, etc., can solve the problem of rapid screening of recombinant bacteria that are difficult to achieve high expression of target proteins, high copy does not necessarily high expression , Screening workload and other problems, to achieve the effect of fast and convenient screening method

Inactive Publication Date: 2012-09-05
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are disadvantages such as cumbersome operation, high cost, and easy gene loss.
Using the LEU2 or URA3 gene of Saccharomyces cerevisiae as a selection marker, although the inorganic medium can be used to exert pressure during the entire fermentation process to avoid the loss of the exogenous gene, its copy number is relatively low, and the operation is cumbersome (medium preparation). Disadvantages such as large quantity and high cost
And sometimes high copy is not necessarily high expression
Generally speaking, the above commonly used screening methods are difficult to achieve rapid screening of recombinant bacteria with high expression of the target protein

Method used

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  • Method for quickly screening yeast with high-expression target proteins
  • Method for quickly screening yeast with high-expression target proteins
  • Method for quickly screening yeast with high-expression target proteins

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Experimental program
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Embodiment 1

[0024] Embodiment 1, acid α-amylase gene as screening marker

[0025] 1. Construction of an expression vector using the acid α-amylase gene as a selection marker

[0026] According to Aspergillus acid α-amylase gene, design primer and carry out PCR amplification, template is the nucleotide sequence of primer used for Aspergillus niger AS 3.795 (purchased from China General Microorganism Culture Collection Management Center) as follows:

[0027] S1: 5'-ATAAGAAT GCGGCCGCGG ATGAGATTATCGACTTCGAG-3',

[0028] S2: 5'-GC TCTAGA ACAGTAACCGACCACTCC-3'.

[0029] The PCR amplification system was 50ul; the amplification program was 94°C for 5min; 94°C for 1min, 56°C for 45sec, 72°C for 2min, 30 cycles; and 72°C for 10min.

[0030] PCR amplification obtained the Aspergillus niger acid α-amylase gene fragment with the NotI enzyme recognition site added to the 5' end and the XbaI enzyme recognition site added to the 3' end. The product was sequenced, and the sequencing results showed ...

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Abstract

The invention discloses a method for quickly screening yeast with high-expression target proteins. In the method, coding genes of acid alpha-amylase are used as screening marker genes. The invention also discloses a carrier which uses the coding genes of the acid alpha-amylase as the screening marker genes. The method for quickly screening the yeast with the high-expression target proteins comprises the following steps: introducing the carrier into the yeast to obtain recombinant strains, culturing the recombinant strains on a solid medium containing soluble starch, and monocloning the recombinant strains around which transparent circles with the diameter of 0.3 to 0.5cm are produced to obtain the yeast with the high-expression target proteins. The method can screen high-expression transformants of the target proteins from macroscopic view; and the screening method is quick and convenient and can be widely used in the production of the target proteins.

Description

technical field [0001] The invention relates to a method for rapidly screening yeast highly expressing a target protein. Background technique [0002] Methanotrophic yeast (referred to as methanol yeast) is an ideal system for expressing foreign genes, especially eukaryotic genes, which has gradually developed in the last decade. This kind of yeast belongs to four genera of Candida, Hansenula, Pichia and Torulopsis, and they can all be cultured in the environment with methanol as the only carbon source and energy source. base growth. Compared with Saccharomyces cerevisiae, methanol yeast has the advantages of stable genetic properties, high expression level, high secretion efficiency and suitable for large-scale fermentation. Hansenula polymorpha (Hansenula) is the leader of this type of yeast, and it is currently recognized internationally as one of the most ideal exogenous gene expression systems. At present, a variety of vegetative mutant strains of Hansenula polymorph...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12R1/645
Inventor 邱并生牛振东宋浩雷
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI