title: Recombinant expression and purification of Ssa1p (Hsp70) from Saccharomyces cerevisiae using Pichia pastoris.
abstract: Heat shock proteins with a molecular mass of 70000 (Hsp70s) are a ubiquitous class of ATP-dependent molecular chaperones involved in the folding of cellular proteins. Sequencing the entire genome of Saccharomyces cerevisiae revealed 14 different genes for Hsp70 proteins in different cellular compartments. Among these 14 Hsp70s, the subclass of Ssa (Ssa1p-Ssa4p) is abundant and essential in the cytosol. Since high yield expression of cytoplasmic Ssa1p is inefficient in Saccharomyces cerevisiae and recombinant expression in E. coli yields low protein levels, we chose Pichia pastoris as the recombinant expression system. In Pichia pastoris, expression levels of Ssa1p are high and Ssa1p is soluble and correctly folded. Also, we present a new protocol for purification of Ssa1p. Previously described purifications include ATP-agarose chromatography leading to Ssa1p partially complexed with ATP. Our optimized purification protocol follows the CiPP strategy (capture, intermediate purification, polishing) avoiding ATP-agarose chromatography, which allows detailed studies on the ATP-dependent Hsp70 functions. We obtained Ssa1p in high purity and 400 times higher quantity compared to previous studies.
authors.affiliation: Institut für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, D-85747, Garching, Germany. harald.wegele@ch.tum.de
authors.forename: Harald
authors.initials: H
authors.lastname: Wegele
authors.forename: Martin
authors.initials: M
authors.lastname: Haslbeck
authors.forename: Johannes
authors.initials: J
authors.lastname: Buchner
mesh_terms: D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000302:isolation & purification; D013058:Mass Spectrometry; D010843:Pichia / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D018840:HSP70 Heat-Shock Proteins; D029701:Saccharomyces cerevisiae Proteins