title: Esterase 2 from Alicyclobacillus acidocaldarius as a reporter and affinity tag for expression and single step purification of polypeptides.
abstract: 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 Alicyclobacillus acidocaldarius. Presence of esterase allows to monitor the expression of fusion proteins spectrophotometrically 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.
authors.affiliation: Laboratorium für Biochemie, Universität Bayreuth, 95440 Bayreuth, Germany.
authors.forename: Yiwei
authors.initials: Y
authors.lastname: Huang
authors.forename: Martin
authors.initials: M
authors.lastname: Humenik
authors.forename: Mathias
authors.initials: M
authors.lastname: Sprinzl
mesh_terms: D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D015951:Factor Xa / Q000737:chemistry; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D010455:Peptides; D011993:Recombinant Fusion Proteins; C047264:sepharose CL 6B; D012685:Sepharose; D004950:Esterases; D015951:Factor Xa