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A method for constructing engineering yeast expressing dual-channel proteins

A technology for protein expression and engineering yeast, which is applied in the field of engineering yeast for dual-channel protein expression, and can solve problems such as inability to be easily interchanged

Active Publication Date: 2021-04-02
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mice are commonly used experimental animals, mouse LFA-1 is highly similar to human protein, but they cannot be easily interchanged with each other

Method used

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  • A method for constructing engineering yeast expressing dual-channel proteins
  • A method for constructing engineering yeast expressing dual-channel proteins
  • A method for constructing engineering yeast expressing dual-channel proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042]Example 1: Two-channel protein expression engineering yeast plasmid vector construction

[0043]Seefigure 2 : To own pCTCON-wGFP plasmid as template, a primer wGFP (5, -ATCGAATTCTACTTCATACATTTTCAAATTAAGATGGCTAGCGTGAGCAAGGGCGAGGAG-3 ,, sequence specific primers plus ECORI site) with a linker and restriction sites wGFP (5, -GTTCGGATCCAGTGATCCCGGCGGCGTTC-3 ,, Sequence specific primers plus BamHI sites) Amplified WGFP fragment (720bp). PCR reaction conditions: 94 ° C predetermitability 3min; 94 ° C 30 sec, 54 ° C 30SEC, 72 ° C for 1 min, 35 cycles; 72 ° C extends for 10 min. The amplified WGFP gene PCR product was inserted between the EcoRI and the BamHi enzyme after the YS2H vector promoter GAL1, screening positive cloning and sequencing, gaining gene WGFP, and we name this plasmid PDV3-intra-WGFP.

[0044]Based on the construction of the PDV3-Intra-WGFP carrier, the applicant is inserted into the mouse LFA-1αL I Domain gene between NCOI and SALI enzyme digestion. . Both the WT and F29...

Embodiment 2

[0049]Example 2: Imported yeast and inducible protein expression of two-channel protein expression engineering yeast carrier

[0050]The above-mentioned recombinant expression carrier vectors encoded WGFP and LFA-1αL I Domain gene (sequence seq ID NO: 1 and SEQ ID NO: 2, 3, 4, 5) are used in classic PEG / LIAC method (ITO H, Fukuda Y) Murata K, ETAL.TRANSFORMATION OF Intact Yeast Cells Treated with Alkali CALLS Treated with Alkali CACions, 1983, 153 (1): 163-168.) Transferred into the yeast cell EBY100 to obtain recombinant yeast cells, cultured the recombination Yeast cells and induced expression of the recombinant vector (specific steps) (for specific steps): hu x, kang s, chen x, et al. Yeast Surface Two-hybrid for quantitative in vivo detection ofprotein-protein interacts via the secretory Pathway [J] .journal The method of reported by Ofbiological Chemistry, 2009,284 (24): 16369-16376, resulting in yeast cells expressing a target protein.

[0051]Specific steps are as follows:

[0052](...

Embodiment 3

[0056]Example 3: Dual Channel Protein Expression Project Yeast Protein Expression Verification

[0057]The recombinant yeast can be inspected by flow cytometry, respectively, and the expression of GFP can be examined by flow cytometry. Idomain expression is subject to Flag tag protein, and it is detected with flow cytometry. Specific steps are as follows:

[0058](1) Suction 3 ul Expresses yeast cells with WGFP and LFA-1αL I Domain protein to add a 96-well V-plate, a negative control, add 100 μl of label buffer to each well to each well. (Formula: PBS + 0.5% BSA + 10mm MGCL2), Mixed back, centrifugation (4 ° C, 3 min, 3000 rpm) to precipitate the cells and go to the supernatant. Joint buffer A, add 20 μl of 10 μl of 10 μg / ml of an anti-mouse anti-FLAG antibody (US Santa Cruz Biotechnology, Santa Cruz Biotechnology) buffer A, 30 ° C oscillated for 30 min.

[0059](2) Add 100 μl of buffer A to wash yeast cells, centrifugal (4 ° C, 3 min, 3000 rpm) to discard the supernatant, then add 20 μl o...

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Abstract

The invention belongs to the technical field of genetic engineering, and in particular relates to a construction method of an engineering yeast of double-channel protein expression. A genetic engineering yeast plasmid vector is constructed; two promoters, namely GAL10 and GAL1, expressing exogenous genes are carried in the plasmid vector, wherein exogenous protein is located to the surface of theyeast by virtue of the promoter GAL10; and protein of the gene, under the control of the promoter GAL1, is expressed in cytoplasm of the yeast. According to the engineering yeast provided by the invention, two different proteins can be conveyed to the surface of the yeast or located into the cytoplasm through different protein expression channels; and the protein on the surface of the yeast is applicable to researches on protein functions, and through intracellular fluorescence intensity, yeast quantity is indicated, so that purposes of being efficient, convenient and economical are achieved,and the construction method is applicable to intracellular protein researches.

Description

Technical field[0001]The present invention belongs to the field of genetic engineering, involving a method of constructing a two-channel protein expression engineering yeast, and the present invention constructs a yeast mass product that carries two promoters GAL10 and GAL1 expressing an exogenous gene, wherein the promoter The gene expression of GAL10 is fused with yeast condensation, since the agglutinium subunit will covalently bind to the agglutinate base of the yeast surface, thereby positioning the exogenous protein to the yeast surface; another startup The gene controlled by the child GAL1 is expressed in the cytoplasmic of yeast. The engineering yeast of the present invention can deliver two different proteins through different protein expression passages to the yeast surface or is positioned in the cellular mass.Background technique[0002]Yeast Surface Explanation (Abbreviations YSD) is a technique that is expressed in yeast in yeast and carries it into a cell surface throug...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/81C12N15/66
CPCC07K14/47C07K2319/02C07K2319/41C07K2319/43C07K2319/60C12N9/2465C12N15/66C12N15/81C12N2800/102C12N2800/60C12Y302/01022
Inventor 胡学博张启云
Owner HUAZHONG AGRI UNIV
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