期刊
ANALYTICAL BIOCHEMISTRY
卷 376, 期 1, 页码 61-72出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2008.01.023
关键词
calspermin; calmodulin; surface plasmon resonance; SPR; kinetics; Arabidopsis
资金
- BBSRC [BB/E002692/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/E002692/1] Funding Source: researchfish
- Biotechnology and Biological Sciences Research Council [BB/E002692/1, BBS/S/K/2004/11180] Funding Source: Medline
We cloned, expressed, and purified a chimeric fusion between a soluble green fluorescent protein (smGFP) and the calmodulin bindingprotein calspermin. We have shown that the fusion protein, labeled smGN, has a K-i in the calmodulin-dependent cyclic nucleotide phosphodiesterase activity assay of 1.97 nM, i.e., 3800 times smaller than that of the commonly used calmodulin inhibitor W7. Association and dissociation rate constants (k(a) and k(d)) and the dissociation equilibrium constant (K-D) of smGN for calmodulin were determined using surface plasmon resonance (SPR). The k(a) = 1.24.10(6) M-1 s(-1), the k(d) = 5.49. 10-3 s-1, and the K-D = 4.42.10(-9) M. We also found that the GFP moiety was important for successfully binding calspermin to the surface of the CM5 flow cell at a sufficiently high concentration for SPR, and that this procedure may be used for SPR analysis of other acidic polypeptides, whose pI <= 4. To determine whether smGN might also bind to other calmodulin-like proteins in a heterologous system, we purified proteins from a plant total cell extract or a plant total protein extract by affinity chromatography against smGN. The purified proteins were identified as calmodulins by sodium dodecyl sulfate-polyacrylamide get electrophoresis and liquid chromatography-tandem mass spectrometry, indicating a high level of specificity. We conclude that the high affinity and specific binding between smGN and calmodulin make it an easily localized recombinant alternative to chemical calmodulin inhibitors. 2008 Elsevier Inc. All rights reserved.
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