title: Disulfide linkages in Plasmodium falciparum plasmepsin-i are essential elements for its processing activity and multi-milligram recombinant production yield.
abstract: 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.
authors.affiliation: Department of Biochemistry and Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors.forename: Sirisak
authors.initials: S
authors.lastname: Lolupiman
authors.affiliation: Department of Medical Technology, Faculty of Allied Health Sciences, Thammasat University, Pathumthani, Thailand.
authors.forename: Pilaiwan
authors.initials: P
authors.lastname: Siripurkpong
authors.affiliation: Department of Biochemistry and Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors.forename: Jirundon
authors.initials: J
authors.lastname: Yuvaniyama
mesh_terms: D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; C089643:plasmepsin