title: Expression, purification, and inhibitory activities of mouse cytotoxic T-lymphocyte antigen-2alpha.
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.
authors.affiliation: Laboratory of Biochemistry and Radiation Biology, Department of Veterinary Sciences, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Kurata
authors.forename: Maki
authors.initials: M
authors.lastname: Hirata
authors.forename: Shoji
authors.initials: S
authors.lastname: Watabe
authors.forename: Masashi
authors.initials: M
authors.lastname: Miyake
authors.forename: Susumu Y
authors.initials: SY
authors.lastname: Takahashi
authors.forename: Yoshimi
authors.initials: Y
authors.lastname: Yamamoto
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000943:Antigens, Differentiation / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology*; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D016415:Sequence Alignment
chemical_list: D000943:Antigens, Differentiation; D015853:Cysteine Proteinase Inhibitors; D011993:Recombinant Fusion Proteins; C102076:cytotoxic T-lymphocyte antigen-2, mouse; D003546:Cysteine Endopeptidases