Open AccessDisulfide Linkages in Plasmodium falciparum Plasmepsin-I Are Essential Elements for Its Processing Activity and Multi-Milligram Recombinant Production Yield
Author(s) -
Sirisak Lolupiman,
Pilaiwan Siripurkpong,
Jirundon Yuvaniyama
Publication year - 2014
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0089424
Subject(s) - plasmodium falciparum , heterologous expression , escherichia coli , yield (engineering) , recombinant dna , enzyme , biochemistry , disulfide bond , heterologous , chemistry , protein disulfide isomerase , biology , malaria , materials science , metallurgy , immunology , gene
Plasmodium falciparum plasmepsin-I (PM-I) has been considered a potential drug target for the parasite that causes fatal malaria in human. Determination of PM-I structures for rational design of its inhibitors is hindered by the difficulty in obtaining large quantity of soluble enzyme. Nearly all attempts for its heterologous expression in Escherichia coli result in the production of insoluble proteins in both semi-pro-PM-I and its truncated form, and thus require protein refolding. Moreover, the yields of purified, soluble PM-I from all reported studies are very limited. Exclusion of truncated semi-pro-PM-I expression in E. coli C41(DE3) is herein reported. We also show that the low preparation yield of purified semi-pro-PM-I with autoprocessing ability is mainly a result of structural instability of the refolded enzyme in acidic conditions due to incomplete formation of disulfide linkages. Upon formation of at least one of the two natural disulfide bonds, nearly all of the refolded semi-pro-PM-I could be activated to its mature form. A significantly improved yield of 10 mg of semi-pro-PM-I per liter of culture, which resulted in 6–8 mg of the mature PM-I, was routinely obtained using this strategy.