,Cloning and Identification of Methionine Synthase Gene from Pichia pastoris

• 作 者: Lan HUANG Dong-Yang LI Shao-Xiao WANG Shi-Ming ZHANG Jun-Hui CHEN Xiang-Fu WU
• 机 构: State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University
• 出 处: 生物化学与生物物理学报（英文版）
• 发表日期: 2005年6期
• 关键词: methionine synthase Pichia pastoris rapid amplification of cDNA end (RACE) funct

Methionine synthase (MS) is grouped into two classes. Class One MS (MetH) and Class Two MS (MetE) share no homology and differ in their catalytic model. Based on the conserved sequences of metE genes from different organisms, a segment of the metE gene was first cloned from Pichia pastoris genomic DNA by PCR, and its 5’ and 3’ regions were further cloned by 5’- and 3’-rapid amplification of cDNA ends (RACE), respectively. The assembled sequence reveals an open reading frame encoding a polypeptide of 768 residues, and the deduced product shares 76% identity with MetE of Saccharomyces cerevisiae. P. pastoris methionine synthase (PpMetE) consists of two domains common to MetEs. The active site is located in the C-terminal domain, in which the residues involved in the interaction of zinc with substrates are conserved. Homologous expression of PpMetE in P. pastoris was achieved, and the heterologous expression of PpMetE in the S. cerevisiae strain XJB3-1D that is MetE-defective restored the growth of the mutant on methionine-free minimal media. The gene sequence has been submitted to GenBank/EMBL/DDBJ under accession No. AY601648.