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purification method for recombinant proteins based on a fusion between the target protein and the c-terminus of calmodulinCalmodulin (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.
Document ID
20040088184
Document Type
Reprint (Version printed in journal)
Authors
Schauer-Vukasinovic, Vesna
(University of Kentucky Lexington, Kentucky 40506-0055, United States)
Deo, Sapna K.
Daunert, Sylvia
Date Acquired
August 21, 2013
Publication Date
July 1, 2002
Publication Information
Publication: Analytical and bioanalytical chemistry
Volume: 373
Issue: 6
ISSN: 1618-2642
Subject Category
Life Sciences (General)
Funding Number(s)
CONTRACT_GRANT: GM 47915
Distribution Limits
Public
Copyright
Other
Keywords
Non-NASA Center
NASA Discipline Life Sciences Technologies