Cloning, soluble expression and mutant activity analysis of lactate dehydrogenase gene from Plasmodium falciparum.
- Author:
Xiao-Ling XU
1
;
Rui-Yi YANG
;
Xue-Qin YANG
;
Li-Ling FENG
;
Qing-Ping ZENG
Author Information
1. Biotechnology Laboratory, Tropical Medicine Institute, Guangzhou University of Chinese Medicine, Guangzhou 510405, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Cloning, Molecular;
Escherichia coli;
genetics;
metabolism;
Inclusion Bodies;
genetics;
metabolism;
L-Lactate Dehydrogenase;
genetics;
metabolism;
Plasmodium falciparum;
enzymology;
genetics;
Protozoan Proteins;
genetics;
metabolism;
Recombinant Fusion Proteins;
genetics;
metabolism;
Solubility
- From:
Chinese Journal of Biotechnology
2007;23(4):593-597
- CountryChina
- Language:Chinese
-
Abstract:
To establish a platform for high throughput screening and in vitro evaluating novel metabolic enzyme-targeted inhibitors towards anti-malarial drugs, a lactate dehydrogenase gene of Plasmodium falciparum (PfLDH) was amplified from the Hainan isolate FCC1/HN. The fusion expression vectors, pGEX-2TK and pET-29a( + ), were utilized to introduce the PfLDH gene into strains of Escherichia coli, BL21 and BL21 (DE3), for over-expression. Consequently, the enzymatic activity of PfLDH was successfully detected in the suspension of lytic bacteria. The PfLDH gene cloned in pGEX-2TK was mainly expressed as inclusion bodies, while the same gene cloned in pET-29a( + ) was nearly expressed in a soluble form of PfLDH, demonstrating the latter vehicle might be more suitable for the large-scale preparation of recombinant PfLDH. Furthermore, according to the electrophoregram of SDS-PAGE and the sequencing data, a series of truncated PfLDH sequences generated randomly from gene amplification were screened and cloned, from which four pre-matured genes with a terminator mutation, PfLDH-delta271, -delta236, -delta167 and -delta53 coding for 45, 80, 149 and 263 amino acid residues, were individually recovered. Through the gene expression and enzymatic activity measurement, the effect of pre-matured terminator mutation on the activity of PfLDH was evaluated, which should pave the way for probing the relationship between structure and function of PfLDH.
