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Purification of Recombinant Proteins Based on the Interaction between a Phenothiazine‐Derivatized Column and a Calmodulin Fusion Tail
Author(s) -
SchauerVukasinovic Vesna,
Daunert Sylvia
Publication year - 1999
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp990058l
Subject(s) - calmodulin , fusion protein , affinity chromatography , phenothiazine , tandem affinity purification , recombinant dna , protein a/g , chemistry , biochemistry , polyacrylamide gel electrophoresis , glutathione s transferase , glutathione , gel electrophoresis , escherichia coli , chromatography , microbiology and biotechnology , biology , enzyme , gene , pharmacology
A method to purify proteins by fusing them to the Ca 2+ ‐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 Ca 2+ . Calmodulin was used as an affinity tail to enable binding of the fusion protein to the phenothiazine column. Removal of Ca 2+ 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).