Purification method for recombinant proteins based on a fusion between the target protein and the C-terminus of calmodulin.
Calmodulin (CaM) was used as an affinity tail to facilitate the purification of the green fluorescent protein (GFP), which was used as a model target protein. The protein GFP was fused to the C-terminus of CaM, and a factor Xa cleavage site was introduced between the two proteins. A CaM-GFP fusion protein was expressed in E. coli and purified on a phenothiazine-derivatized silica column. CaM binds to the phenothiazine on the column in a Ca(2+)-dependent fashion and it was, therefore, used as an affinity tail for the purification of GFP. The fusion protein bound to the affinity column was then subjected to a proteolytic digestion with factor Xa. Pure GFP was eluted with a Ca(2+)-containing buffer, while CaM was eluted later with a buffer containing the Ca(2+)-chelating agent EGTA. The purity of the isolated GFP was verified by SDS-PAGE, and the fluorescence properties of the purified GFP were characterized.
Schauer-Vukasinovic
Vesna
V
Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055, USA.
Deo
Sapna K
SK
Daunert
Sylvia
S
D001483:Base Sequence; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015951:Factor Xa / Q000302:isolation & purification; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000302:isolation & purification; D010446:Peptide Fragments / Q000302:isolation & purification; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods
D002147:Calmodulin; D017931:DNA Primers; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D015951:Factor Xa
NASA Discipline Life Sciences Technologies; Non-NASA Center