Purification method for recombinant proteins based on a fusion between the target protein and the C-terminus of calmodulin

Vesna Schauer-Vukasinovic, Sapna K. Deo, Sylvia Daunert

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

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 Ca2+-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 Ca2+-containing buffer, while CaM was eluted later with a buffer containing the Ca2+-chelating agent EGTA. The purity of the isolated GFP was verified by SDS-PAGE, and the fluorescence properties of the purified GFP were characterized.

Original languageEnglish (US)
Pages (from-to)501-507
Number of pages7
JournalAnalytical and bioanalytical chemistry
Volume373
Issue number6
DOIs
StatePublished - Jan 1 2002
Externally publishedYes

Keywords

  • Affinity purification
  • C-terminal fusion
  • Calmodulin
  • Factor Xa cleavage
  • GFP

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Analytical Chemistry

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