Expression and Identification of CP4-EPSPS Gene in Pichia pastoris
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Abstract:
Constructing the genetically modified (GM) Pichia pastoris stains is the key step forestablishing a new method to assess the safety of foreign protein . Based on the research model of the 5-enolpyruvyl shikinmate-3-phosphate synthase derived from Agrobacterium CP4 strain (CP4-EPSPS), the cp4-epsps gene was chemically synthesized after optimizing thecodon usage bias. The codon adaption index (CAI) of optimized sequence was 0.85, and the GC content of optimized sequence was 52%. Cp4-epsps gene was amplified by PCR and cloned to the expression vector pPICZb of Pichia pastoris GS115 through homologous end-joining, and the recombinant vector pPICZb-EPSPS was constructed. The correct recombinant vector was transferred into GS115 strain through electroporation, and four positive strains were screened through the right site via PCR. Then, those positive strains were induced by 0.5% methanol for four days at 28 ℃, 250~300 r/min. Finally, the expression of cp4-epsps in GS115 was identified from CP4-EPSPS monoclonal antibody and C-MYC monoclonal antibody by SDS-PAGE and western blotting. The results showed that the target gene was expressed in GS115 strain and MW of protein was 50 ku, which coincided with the theoretical results. In conclusion, the construction of transgenic Pichia pastoris GS115 strains is beneficial for the safety assessment of transgenic CP4-EPSPS synthase in GM crops.
