Cloning, soluble expression and mutant activity analysis of lactate dehydrogenase gene from Plasmodium falciparum / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 593-597, 2007.
Article
in Chinese
| WPRIM
| ID: wpr-327981
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Plasmodium falciparum
/
Solubility
/
Recombinant Fusion Proteins
/
Protozoan Proteins
/
Inclusion Bodies
/
Cloning, Molecular
/
Escherichia coli
/
Genetics
/
L-Lactate Dehydrogenase
/
Metabolism
Limits:
Animals
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2007
Type:
Article
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