Using capillary electrophoresis with laser-induced fluorescence to study the interaction of green fluorescent protein-labeled calmodulin with Ca2+- and calmodulin-binding protein.
A separation using capillary electrophoresis with laser-induced fluorescence (CE-LIF) was applied to the study of green fluorescent protein tagged calmoldulin (GFP-CaM) that was expressed from Escherichia coli and purified with Ni(2+)-nitrilotriacetate (Ni-NTA) resin column. It was found that GFP-CaM not only has good fluorescence properties under various conditions similar to GFP, but also retains its calcium-binding ability as the native CaM. GFP-CaM was separated and detected by CE-LIF within 10 min with a limit-of-detection (LOD) of 2 x 10(-10) M for an injection volume of 3 nl, higher than that of common chemical fluorescent-tagged protein method. The results indicated that, as a fluorescence probe, GFP could overcome the drawback of inefficient derivatization of chemical fluorescence probes. The interaction between the GFP-CaM and Ca(2+) was studied in detail using affinity capillary electrophoresis with laser-induced fluorescence and the dissociation constant (K(d)) between GFP-CaM and Ca(2+) was determined to be 1.2 x 10(-5), which is in good agreement with the literature values of untagged CaM (10(-6) to 10(-5)M) obtained by conventional method. As a preliminary application, the interaction between GFP-CaM and OsCBK was also investigated. The method makes it possible to screen the trace amounts of target proteins in crude extracts interacting with CaM under physiological conditions.
Zhang
Jian-Feng
JF
Key Laboratory of MOE for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China.
Ma
Li
L
Liu
Xin
X
Lu
Ying-Tang
YT
D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D017931:DNA Primers; D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry; D007834:Lasers; D011485:Protein Binding
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D002118:Calcium