TY - JOUR
T1 - Recombinant expression and purification of Ssa1p (Hsp70) from Saccharomyces cerevisiae using Pichia pastoris
AU - Wegele, Harald
AU - Haslbeck, Martin
AU - Buchner, Johannes
N1 - Funding Information:
We would like to thank Elizabeth Craig (University of Wisconsin, Madison, WI, USA) for the kind gift of Ssa antibody. We gratefully acknowledge the experimental help of Holger Grallert and thank Helmut Krause for performing the mass spectroscopy. We also would like to thank Thomas Scheibel and Stefan Walter for inspiring discussions and help with the manuscript. Harald Wegele was supported by fellowships of the Studienstiftung des deutschen Volkes and the Fonds der Chemischen Industrie.
PY - 2003/3/25
Y1 - 2003/3/25
N2 - Heat shock proteins with a molecular mass of 70 000 (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.
AB - Heat shock proteins with a molecular mass of 70 000 (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.
KW - ATPase
KW - Hsp70
KW - Purification
KW - Ssa1p
UR - http://www.scopus.com/inward/record.url?scp=0037465664&partnerID=8YFLogxK
U2 - 10.1016/S1570-0232(02)00724-9
DO - 10.1016/S1570-0232(02)00724-9
M3 - Article
C2 - 12651006
AN - SCOPUS:0037465664
SN - 1570-0232
VL - 786
SP - 109
EP - 115
JO - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
JF - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
IS - 1-2
ER -