Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 54, Issue 1, Pages 94-100Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2007.02.005
Keywords
esterase; reporter enzyme; affinity tag; trifluoromethyl-alkyl-ketone
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A novel dual function (reporter and affinity) tag system has been developed. Expression vectors have been constructed to express polypeptides in Escherichia coli cells as C-terminal fusions with esterase 2, a 34-kDa protein from Alicyclobacillits acidocaldarius. Presence of esterase allows to monitor the expression of fusion proteins spectrophotornetrically or by activity staining in the polyacrylamide gels. The fusion proteins can be purified from crude bacterial extracts under non-denaturing conditions by one step affinity chromatography on Sepharose CL-6B immobilized trifluoromethyl-alkyl-ketone. The esterase carrier can be cleaved from fusion proteins by digestion with amino acid sequence-specific proteases blood coagulation factor Xa. The system has been used successfully for the expression and purification of polypeptides from different prokaryotic and eukaryotic organisms. (c) 2007 Elsevier Inc. All rights reserved.
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