Cloning，Soluble Expression and Mutant Activity Analysis of Lactate Dehydrogenase Gene from Plasmodium falciparum

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-Δ271,-Δ236,-Δ167 and -Δ53 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.

Key words:PfLDH，fusion expression，terminator mutation，enzymatic activity