Purification of recombinant proteins based on the interaction between a phenothiazine-derivatized column and a calmodulin fusion tail

Vesna Schauer-Vukasinovic, Sylvia Daunert

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

A method to purify proteins by fusing them to the Ca2+-dependent protein calmodulin is described by using glutathione-S-transferase (GST) from Schistosoma japonicum as a model. Glutathione-S-transferase was genetically fused to calmodulin (CaM). The designed GST-CaM fusion protein has a selective factor Xa cleavage site located between the C-terminus of GST and the N-terminus of CaM. The recombinant fusion protein was expressed in Escherichia coli, and the crude cell extract was loaded onto a phenothiazine affinity column in the presence of Ca2+. Calmodulin was used as an affinity tail to enable binding of the fusion protein to the phenothiazine column. Removal of Ca2+ with a calcium-complexing solution causes elution of the fusion protein. The GST-CaM fusion protein was then digested with factor Xa, and the target protein GST was isolated. The purity of the isolated GST was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Original languageEnglish (US)
Pages (from-to)513-516
Number of pages4
JournalBiotechnology Progress
Volume15
Issue number3
DOIs
StatePublished - May 1999
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology

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