title: Codon optimisation improves the expression of Trichoderma viride sp. endochitinase in Pichia pastoris.
abstract: The mature cDNA of endochitinase from Trichoderma viride sp. was optimised based on the codon bias of Pichia pastoris GS115 and synthesised by successive PCR; the sequence was then transformed into P. pastoris GS115 via electroporation. The transformant with the fastest growth rate on YPD plates containing 4 mg/mL G418 was screened and identified. This transformant produced 23.09 U/mL of the recombinant endochitinase, a 35% increase compared to the original strain bearing the wild-type endochitinase cDNA. The recombinant endochitinase was sequentially purified by ammonia sulphate precipitation, DE-52 anion-exchange chromatography and Sephadex G-100 size-exclusion chromatography. Thin-layer chromatography indicated that the purified endochitinase could hydrolyse chito-oligomers or colloidal chitin to generate diacetyl-chitobiose (GlcNAc)₂ as the main product. This study demonstrates (1) a means for high expression of Trichoderma viride sp. endochitinase in P. pastoris using codon optimisation and (2) the preparation of chito-oligomers using endochitinase.
authors.affiliation: College of Food Science and Biotechnology, Zhejiang Gongshang University, 149 Jiaogong Road, Hangzhou 310035, Zhejiang Province, People's Republic of China.
authors.forename: Ping
authors.initials: P
authors.lastname: Yu
authors.forename: Yuan
authors.initials: Y
authors.lastname: Yan
authors.forename: Qing
authors.initials: Q
authors.lastname: Gu
authors.forename: Xiangyang
authors.initials: X
authors.lastname: Wang
mesh_terms: D001483:Base Sequence; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002855:Chromatography, Thin Layer; D003062:Codon; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D014242:Trichoderma / Q000235:genetics* / Q000378:metabolism
chemical_list: D003062:Codon; D018076:DNA, Complementary; D005656:Fungal Proteins; D011994:Recombinant Proteins; D002688:Chitinases