[Cloning, soluble expression and mutant activity analysis of lactate dehydrogenase gene from Plasmodium falciparum].
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.
Xu
Xiao-Ling
XL
Biotechnology Laboratory, Tropical Medicine Institute, Guangzhou University of Chinese Medicine, Guangzhou 510405, China.
Yang
Rui-Yi
RY
Yang
Xue-Qin
XQ
Feng
Li-Ling
LL
Zeng
Qing-Ping
QP
D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012995:Solubility