Synthesis of Drosophila melanogaster acetylcholinesterase gene using yeast preferred codons and its expression in Pichia pastoris.
To improve the expression level of recombinant Drosophila melanogaster AChE (R-DmAChE) in Pichia pastoris, the cDNA of DmAChE was first optimized and synthesized based on the preferred codon usage of P. pastoris. The synthesized AChE cDNA without glycosylphosphatidylinositol (GPI) signal peptide sequence was then ligated to the P. pastoris expression vector, generating the plasmid pPIC9K/DmAChE. The linearized plasmid was homologously integrated into the genome of P. pastoris GS115 via electrotransformation. Finally seven transformants with high expression level of R-DmAChE activity were obtained. The highest production of R-DmAChE in shake-flask culture after 5-day induction by methanol was 718.50 units/mL, which was about three times higher than our previous expression level of native DmAChE gene in P. pastoris. Thus, these new strains with the ability to secret R-DmAChE in the medium could be used for production of R-DmAChE to decrease the cost of the enzyme expense for rapid detection of organophosphate and carbamate insecticide residues.
Wu
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Bor Luh Food Safety Center, Key Laboratory of Microbial Metabolism, Ministry of Education, School of Life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, PR China.
Chen
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HD
Tang
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Ye
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Liu
W J
WJ
Jia
H Y
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Zhang
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D000110:Acetylcholinesterase / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D003062:Codon; D017931:DNA Primers; D018076:DNA, Complementary; D004331:Drosophila melanogaster / Q000201:enzymology*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics
D003062:Codon; D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D000110:Acetylcholinesterase