title: Utility of (His)6 tag for purification and refolding of proplasmepsin-2 and mutants with altered activation properties.
abstract: Plasmepsin-2 is a malarial aspartic proteinase that has been implicated in the initial steps of hemoglobin degradation in parasites and thus represents an attractive antimalarial target. Escherichia coli expressed proplasmepsin-2 is capable of activation at acidic pH by autocatalytic cleavage of the pro part region, which results in products of different length. We designed a 10-amino-acid deletion in the pro part region that allows faster generation of homogeneous enzyme upon activation. Incorporation of a (His)6 tag onto the N-terminus of the pro part enables on-column refolding of proplasmepsin-2 and simplifies proenzyme purification and pro part separation after activation. The proposed purification procedure results in highly pure and easily crystallizable enzyme.
authors.affiliation: SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201, USA. sgulnik@aol.com
authors.forename: Sergei V
authors.initials: SV
authors.lastname: Gulnik
authors.forename: Elena I
authors.initials: EI
authors.lastname: Afonina
authors.forename: Elena
authors.initials: E
authors.lastname: Gustchina
authors.forename: Betty
authors.initials: B
authors.lastname: Yu
authors.forename: Abelardo M
authors.initials: AM
authors.lastname: Silva
authors.forename: Young
authors.initials: Y
authors.lastname: Kim
authors.forename: John W
authors.initials: JW
authors.lastname: Erickson
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D009154:Mutation; D010961:Plasmodium / Q000378:metabolism*; D017510:Protein Folding; D011499:Protein Processing, Post-Translational
chemical_list: D004792:Enzyme Precursors; D006639:Histidine; D016282:Aspartic Acid Endopeptidases; C116755:proplasmepsin II