Three-step chromatographic purification of Cpr6, a cyclophilin from Saccharomyces cerevisiae.
Cyclophilins constitute a group of peptidyl-prolyl cis-trans isomerases (PPIs), known to be involved in protein folding. Because of their ability to bind the immunosuppresant drug Cyclosporin A (CsA), they are also called immunophilins. Immunophilins, which exhibit a relative molecular mass higher than 40 000, are further found in complex with Hsp90, a major cytosolic molecular chaperone. The present work describes a three-step chromatographic purification of recombinant Cpr6, a cyclophilin from Saccharomyces cerevisiae. The cDNA of Cpr6 was cloned into a pRSET A-plasmid with an N-terminal 6 x histidine-tag (his-tag) and transformed into the BL21[DE3]pLysS strain. After collection of the bacterial material and lysis of the cells the cell lysate was centrifuged and loaded onto a metal chelating column. After extensive washing the protein was eluted with a step gradient from 20 to 250 mM imidazol. The pooled protein was dialysed against ethylenedinitrilo tetraacetic acid (EDTA)-buffer, and loaded onto a strong anion-exchanger. Cpr6 containing fractions were then, in a last step, loaded onto a gel permeation chromatography column. The purity of the resulting protein was measured by silver stained sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and, additionally, as Cpr6 does not contain tryptophan residues by tryptophan residue titration. Based on a standard curve the content of contaminating tryptophan residues in the purified protein solution was determined. A typical yield of 1 mg pure protein per g of wet cells was achieved with the described procedure.
Mayr
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Institut für Organische Chemie und Biochemie, Technische Universität München, Garching, Germany. christian.mayr@biologie.uni-regensburg.de
D002352:Carrier Proteins / Q000302:isolation & purification*; D002853:Chromatography, Liquid / Q000379:methods*; D000081406:Peptidyl-Prolyl Isomerase F; D021983:Cyclophilins; D004591:Electrophoresis, Polyacrylamide Gel; D019696:Peptidylprolyl Isomerase / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D000097322:Peptidyl-Prolyl Isomerase D
D002352:Carrier Proteins; D000081406:Peptidyl-Prolyl Isomerase F; D021983:Cyclophilins; D019696:Peptidylprolyl Isomerase; D011994:Recombinant Proteins; C000631444:CPR6 protein, S cerevisiae; D000097322:Peptidyl-Prolyl Isomerase D