Expression and characterization of a recombinant cysteine proteinase of Leishmania mexicana.
A major cysteine proteinase (CPB) of Leishmania mexicana, that is predominantly expressed in the form of the parasite that causes disease in mammals, has been overexpressed in Escherichia coli and purified from inclusion bodies to apparent homogeneity. The CPB enzyme, CPB2.8, was expressed as an inactive pro-form lacking the characteristic C-terminal extension (CPB2.8DeltaCTE). Pro-region processing was initiated during protein refolding and proceeded through several intermediate stages. Maximum enzyme activity accompanied removal of the entire pro-region. This was facilitated by acidification. Purified mature enzyme gave a single band on SDS/PAGE and gelatin SDS/PAGE gels, co-migrated with native enzyme in L. mexicana lysates, and had the same N-terminal sequence as the native enzyme. The procedure yielded >3.5 mg of active enzyme per litre of E. coli culture.
Sanderson
S J
SJ
Division of Infection and Immunity, Joseph Black Building, Institute of Biomedical and Life Sciences, University of Glasgow, University Avenue, Glasgow G12 8QQ, UK. s.sanderson@bio.gla.ac.uk
Pollock
K G
KG
Hilley
J D
JD
Meldal
M
M
Hilaire
P S
PS
Juliano
M A
MA
Juliano
L
L
Mottram
J C
JC
Coombs
G H
GH
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli; D006801:Humans; D002479:Inclusion Bodies; D007700:Kinetics; D007894:Leishmania mexicana / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases