http://www.abbs.info      E-mail: [email protected]

ISSN 1672-9145                                                Acta Biochim Biophys Sin 2005, 37(6): 371–378                                                   CN 31-1940/Q

Cloning and Identification of Methionine Synthase Gene from Pichia pastoris

Lan HUANG1, Dong-Yang LI2, Shao-Xiao WANG2*, Shi-Ming ZHANG2&, Jun-Hui CHEN1, and Xiang-Fu WU2

 

1 State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing 210093, China;

2 Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

 

Abstract        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.

 

Key words        methionine synthase; Pichia pastoris; rapid amplification of cDNA end (RACE); functional complementation

 

 

 

 

-----------------

Received: January 9, 2005        Accepted: March 31, 2005

This work was supported by a grant from the National Natural Science Foundation of China (No. 30200002)

& Present address: Weis Center for Research, Geisinger Health System, Danville, PA 17822, USA

*Corresponding author: Tel, 86-21-54921085; Fax, 86-21-54921083; E-mail, [email protected]