| 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
|
|
WOS Category
|
BIOCHEMICAL RESEARCH METHODS
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
| Resource |
| Human and Animal Cells |
P-815(RCB1167) |
