RRC ID 1270
Author Kurata M, Hirata M, Watabe S, Miyake M, Takahashi SY, Yamamoto Y.
Title Expression, purification, and inhibitory activities of mouse cytotoxic T-lymphocyte antigen-2alpha.
Journal Protein Expr Purif
Abstract Cytotoxic T-lymphocyte antigen-2 (CTLA-2) is a novel cysteine proteinase inhibitor. The protein sequence is homologous to the proregion of mouse cathepsin L. Here, we report the expression, purification, and characterization of recombinant CTLA-2 (CTLA-2alpha). CTLA-2alpha was cloned into the pET16b vector and the plasmid was transformed into Escherichia coli strain BL21 (DE3) pLysS. The recombinant CTLA-2alpha was highly expressed and purified by His-Bind affinity chromatography, Factor Xa digestion, and hydrophobic chromatography. Throughout these procedures, 3mg recombinant CTLA-2alpha was obtained from 450 ml of bacterial culture medium. The purified protein exhibited inhibitory activities towards certain cysteine proteinases and was properly refolded, as indicated by circular dichroism spectroscopy. Recombinant CTLA-2alpha fully inhibited Bombyx cysteine proteinase (BCP) (overall Kd (Ki*) = 0.23 nM) and and cathepsin L (overall Kd (Ki*) = 0.38 nM). Inhibition of cathepsin H ( Ki = 86 nM) and papain ( Ki = 560 nM) was much weaker, while inhibition of cathepsin B was negligible ( Ki > 1 microM). Our results indicate that mouse CTLA-2alpha is a selective inhibitor of the cathepsin L-like cysteine proteinases.
Volume 32(1)
Pages 119-25
Published 2003-11-1
DOI 10.1016/S1046-5928(03)00222-5
PII S1046592803002225
PMID 14680948
MeSH Amino Acid Sequence Animals Antigens, Differentiation / chemistry Antigens, Differentiation / genetics Antigens, Differentiation / isolation & purification* Antigens, Differentiation / pharmacology* Chromatography, Affinity Circular Dichroism Cloning, Molecular Cysteine Endopeptidases / metabolism Cysteine Proteinase Inhibitors / chemistry Cysteine Proteinase Inhibitors / isolation & purification* Cysteine Proteinase Inhibitors / pharmacology* Mice Molecular Sequence Data Protein Binding Recombinant Fusion Proteins / genetics Recombinant Fusion Proteins / isolation & purification Recombinant Fusion Proteins / pharmacology Sequence Alignment
IF 1.513
Times Cited 21
Human and Animal Cells P-815(RCB1167)