Three-step chromatographic purification of Cpr6, a cyclophilin from Saccharomyces cerevisiae

Cyclophilins constitute a group of peptidyl-prolyl cis-trans isomerases (PP Is), known to be involved in protein folding. Because of their ability to b ind the immunosuppressant drug Cyclosporin A (CsA), they are also called im munophilins. Immunophilins, which exhibit a relative molecular mass higher than 40 000, are further found in complex with Hsp90, a major cytosolic mol ecular chaperone. The present work describes a three-step chromatographic p urification of recombinant Cpr6, a cyclophilin from Saccharomyces cerevisia e. 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. A fter collection of the bacterial material and lysis of the cells the cell l ysate was centrifuged and loaded onto a metal chelating column. After exten sive washing the protein was eluted with a step gradient from 20 to 250 mM imidazol. The pooled protein was dialysed against ethylenedinitrilo tetraac etic acid (EDTA)-buffer, and loaded onto a strong anion-exchanger. Cpr6 con taining fractions were then, in a last step, loaded onto a gel permeation c hromatography column. The purity of the resulting protein was measured by s ilver stained sodium dodecyl sulphate-polyacrylamide gel electrophoresis (S DS-PAGE) and, additionally, as Cpr6 does not contain tryptophan residues by tryptophan residue titration. Based on a standard curve the content of con taminating tryptophan residues in the purified protein solution was determi ned. A typical yield of 1 mg pure protein per g of wet cells was achieved w ith the described procedure. (C) 2000 Elsevier Science B.V. All rights rese rved.