Over-production of lactate dehydrogenase from Plasmodium falciparum opens a route to new antimalarials

被引:23
作者
Turgut-Balik, D [1 ]
Shoemark, DK
Moreton, KM
Sessions, RB
Holbrook, JJ
机构
[1] Univ Firat, Fac Sci & Arts, Dept Biol, Elazig, Turkey
[2] Univ Bristol, Sch Med Sci, Dept Biochem, Bristol BS8 1TD, Avon, England
[3] Univ Bristol, Sch Med Sci, Mol Recognit Ctr, Bristol BS8 1TD, Avon, England
关键词
APAD; lactate dehydrogenase; malaria; Plasmodium falciparum; Shine-Dalgarno;
D O I
10.1023/A:1010555803606
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Over-production of lactate dehydrogenase (PfLDH) from Plasmodium falciparum from E. coli TG2 cells transformed with a pKK223-3 plasmid containing the wild type gene isolated by Bzik DJ, Fox BA, and Gonyer K (1993) Mol. Biochem. Parasit. 59, 155-166, gave mostly an inactive protein after isolation. Sequencing the N-terminus of the over-produced protein showed that the major product commenced at an internal methionine. Truncation of the protein occurred due to the inappropriate priming from a Shine-Dalgarno (SD) sequence upstream of Met 35. Silent mutations of this SD sequence to remove the purine-rich region allowed over-production of the full length PfLDH up to 15 mg protein l(-1) broth. The purified protein exhibited biochemical properties of an authentic LDH enzyme. However, high activity with 3-acetylpyridine adenine dinucleotide as well as with the natural cofactor, NAD, was also observed. The high-resolution X-ray structure obtained from the recombinant enzyme has provided the opportunity for the development of inhibitors specific to PfLDH.
引用
收藏
页码:917 / 921
页数:5
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