A method for high-efficiency expression of carboxypeptidase y derived from Radiomucor elegans
A technology of carboxypeptidase and co-expression, which is applied in the field of high-efficiency expression of carboxypeptidase Y derived from Mucor radiata, can solve the problem that carboxypeptidase Y is rarely reported, and the expression level of carboxypeptidase Y protein is not high, which is not suitable for industrial applications etc.
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Embodiment 1
[0032] Embodiment 1 Construction of recombinant plasmid pPICZα-procpy
[0033] The primers were designed with the laboratory-preserved plasmid pPIC9K-procpy, and the EcoRI restriction site and protective base were introduced upstream, the Not I restriction site and protective base were introduced downstream, and the His tag was introduced at the C-terminal.
[0034] Using pPIC9K-procpy as a template, the full-length procpy gene containing EcoR I, Not I restriction sites and C-terminal His tag was obtained by PCR. The amplified procpy gene product was digested with EcoR I and Not I and connected to pPICZα digested with the same restriction endonuclease to obtain the recombinant plasmid pPICZα-procpy, transformed into Escherichia coli JM109, and sequenced to check whether the sequence was correct .
Embodiment 2
[0035] Example 2 Construction of recombinant Pichia pastoris GS115 / pPICZα-procpy
[0036] The recombinant plasmid pPICZα-procpy with the correct sequence was successfully constructed, digested with the restriction endonuclease Sac I, set the electroporation parameters: 1500v, 400Ω, 4-6ms, electrotransformed Pichia pastoris GS115, and coated with zeocin 100μg / mL Place the YPD plate in an incubator at 28°C for 2-3 days to screen for recombinant strains.
Embodiment 3
[0037] Example 3 Screening of High Copy Recombinant Strains
[0038] Put the transformant on the YPD plate containing zeocin 100 μg / mL, use a sterilized pipette tip to spot on the YPD plate containing zeocin 600, 1000, 1800 μg / mL in sequence, and place it in an incubator at 28°C for 2 -3 days, transformants were screened. The recombinant strain that can grow on the YPD plate containing zeocin 100 μg / mL but cannot grow on the YPD plate containing zeocin 600 μg / mL is named zeocin-100. In this way, zeocin-600, zeocin-1000, zeocin- 1800.
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