Method for improving secretion or surface display expression of heterologous protein in saccharomyces cerevisiae

A heterologous protein, surface display technology, applied in the field of recombinant Saccharomyces cerevisiae strains and its construction, to achieve the effect of improving production costs

Pending Publication Date: 2021-10-12
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the research on improving the surface display and extracellular secretion expression of heterologous proteins in Saccharomyces cerevisiae through the modification of the secretory pathway has been relatively in-depth, there are relatively few studies on other influencing factors.

Method used

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  • Method for improving secretion or surface display expression of heterologous protein in saccharomyces cerevisiae
  • Method for improving secretion or surface display expression of heterologous protein in saccharomyces cerevisiae
  • Method for improving secretion or surface display expression of heterologous protein in saccharomyces cerevisiae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Construction of cellulase β-glucosidase recombinant plasmid

[0038] Taking the cellulase β-glucosidase (β-glucosidase, BGL1) derived from Saccharomycopsis fibuligera as an example, the function of β-glucosidase is to hydrolyze cellobiose to generate glucose. The BGL1 sequence was used as a template, and primers 1 and 2 / 3 / 4 were used for three rounds of PCR amplification to obtain the β-glucosidase fragment 1 containing the V5 tag and connecting sequence. Template amplifies SED1 surface display fragment 2, and then amplifies plasmid backbone fragment 3 with primer 7 and primer 8, and assembles fragment 1, fragment 2, and fragment 3 by the Gibson Assembly method to construct a surface-displayed recombinant plasmid TC001 (such as figure 1 A); Utilize primer 1 and primer 2 / 9 to amplify the secreted β-glucosidase fragment 4, and fragment 3 and fragment 4 are assembled by Gibson Assembly method to construct recombinant plasmid TC002 (such as figure 1 B). Recombin...

Embodiment 2

[0049] Example 2 Preparation of recombinant Saccharomyces cerevisiae library of BGL1 display type and secretory expression

[0050] The obtained recombinant plasmids TC001 and TC002 were respectively transformed into the Saccharomyces cerevisiae knockout library (commercialized) that inactivated cell wall-related proteins, and the inactivated cell wall-related proteins were DFG5, YPS7, TIR1, FKS1, NPP1, LDB17, CCW12, SHE10 , CWP2 and PRY3. The specific steps are as follows: Pick the yeast into 96-well deep-well plates containing 300 μL YEPD medium, activate at 30°C for 36 h, transfer to 500 μL YEPD medium, and dilute 20 times to make the initial OD600 about 0.2, Cultivate at 30°C until the OD600 is between 0.7-1.0, centrifuge at 5000rpm for 5min to remove the YEPD medium, resuspend the cell pellet with 500μL sterile water, centrifuge at 5000rpm for 5min to remove the supernatant, then resuspend the cell pellet with 500μL 100mM LiAC, centrifuge at 5000rpm for 5min Remove the s...

Embodiment 3

[0051] Example 3 Screening of cell wall elements

[0052] In order to screen cell wall elements that can increase the expression of heterologous proteins in Saccharomyces cerevisiae, each unit yeast in the recombinant S. Activation in a 96-well deep-well plate containing 300 μL SC-URA liquid medium for 36 hours, and then diluted 20 times in 600 μL SC-URA liquid medium (1.7g / L amino-free yeast nitrogen source, 5g / L ammonium sulfate, 20g / L D-glucose, 190mg / L arginine, 52mg / L tyrosine, 108mg / L methionine, 290mg / L isoleucine, 440mg / L lysine, 200mg / L phenylalanine, 400mg / L L aspartic acid, 1260mg / L glutamic acid, 380mg / L valine, 220mg / L threonine, 400mg / L leucine, 130mg / L glycine, 40mg / L tryptophan and 140mg / L group Amino acid) in a 96-well deep-well plate, cultured at 30°C for 24 hours to obtain a culture solution containing the heterologous protein BGL1, and carried out the next step of enzyme activity determination. First, the standard curve of the substrate pNP was drawn, an...

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Abstract

The invention relates to a method for improving secretion or surface display expression of a heterologous protein in saccharomyces cerevisiae, and particularly discloses a method for simultaneously improving extracellular secretion and surface display expression of the heterologous protein in the saccharomyces cerevisiae. The method comprises the following step of performing surface display or secretory expression of the heterologous protein by taking one or more cell wall related protein inactivated saccharomyces cerevisiae cells or one or more cell wall related protein gene knockout saccharomyces cerevisiae cells as host cell(s). A method for the surface display or secretory expression of the heterologous protein being beta-glucosidase comprises the following steps: (1) preparing extracellular secretory recombinant plasmids; and (2) transforming the recombinant plasmids into the saccharomyces cerevisiae cell disclosed by the invention, and conducting culture and a enzyme activity test.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a recombinant Saccharomyces cerevisiae strain and a construction method thereof. Background technique [0002] Heterologous proteins include protein drugs and industrial enzymes, and their application research in the fields of medicine, bulk chemical products, and energy has attracted much attention in recent years. Saccharomyces cerevisiae, as an important eukaryotic model organism, is widely used as a chassis cell for the secretion and expression of recombinant proteins. Compared with prokaryotic Escherichia coli (Escherichia coli), it has a protein secretion pathway similar to higher organisms and a relatively complete post-translational modification process, which is conducive to maintaining the natural activity and function of eukaryotic secreted proteins; pastoris), Saccharomyces cerevisiae is an internationally recognized food-grade safety-grade microorganism that ...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/66C12N1/19C12R1/865
CPCC12N15/81C12N15/66C12N9/2402C12Y302/01021
Inventor 汤红婷罗小舟陈南柱
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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