期刊
ANALYTICAL BIOCHEMISTRY
卷 345, 期 2, 页码 214-226出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2005.06.037
关键词
surface plasmon resonance; cyclophilin A; cyclosporin A; competition binding assay
资金
- Biotechnology and Biological Sciences Research Council [BB/D006171/1] Funding Source: researchfish
- Biotechnology and Biological Sciences Research Council [BB/D006171/1] Funding Source: Medline
- Wellcome Trust Funding Source: Medline
A simple protocol for generating a highly stable and active surface plasmon resonance (SPR) sensor surface of recombinant human hexahistidine cyclophilin A (His-CypA) is described. The sensor surface was sensitive and stable enough to allow, for the first time, the screening and ranking of several novel small-molecule (M-r similar to 250-500 Da) ligands in a competition binding assay with cyclosporin A (CsA). It also allowed us to accurately determine the kinetic rate constants for the interaction between His-CypA and CsA. His-CypA was first captured on a Ni2+-nitrilotriacetic acid (NTA) sensor chip and was then briefly covalently stabilized, coupling via primary amines. The significant baseline drift observed due to dissociation of weakly bound His-CypA from the Ni2+- NTA moiety was eliminated, resulting in a surface that was stable for at least 36 h. In addition, immobilized protein activity levels were high, typically between 85 and 95%, assayed by the interaction between His-CypA and CsA. The mean equilibrium dissociation constant for CsA (K-dCsA) binding to the immobilized His-CypA was 23 +/- 6 nM, with on and off rates of 0.53 +/- 0.1 mu M-1 s(-1) and 1.2 +/- 0.1 (x 10(-2)) s(-1), respectively. These values agree well with the values for the corresponding binding constants determined from steady-state and kinetic fluorescence titrations in solution. (C) 2005 Elsevier Inc. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据