Overexpression, purification, and characterization of yeast cyclophilins A and B.
Two isoforms of yeast cyclophilins, yCyPA and yCyPB, have been subcloned, expressed in Escherichia coli, and purified to homogeneity. The full-length (163-amino acid) yeast CyPA was easily expressed and purified; however, only a genetically truncated, 186-residue form of yCyPB lacking a putative 20-amino acid signal sequence could be purified. Each yeast cyclophilin isoform is a peptidyl-prolyl isomerase, inhibitable by the immunosuppressive drug CsA (IC50's of 40 +/- 8 nM and 101 +/- 14 nM at 18 nM concentrations of yCyPA and yCyPB, respectively). Polyclonal antibodies raised against recombinant yCyPA detected native yCyPA in yeast cell extracts by both immunoprecipitation and Western blot analysis. However, polyclonal antibodies raised against recombinant yCyPB detected no native yCyPB in yeast cell extracts by Western blot analysis; small amounts of yCyPB were found in the culture broth, suggesting secretion extracellularly of this isoform. Northern analysis indicated that both yCyPA mRNA and yCYPB mRNA (at a much lower level) were detectable in cell-free extracts. Characterization of the yeast cyclophilin proteins demonstrated that their catalytic properties and sensitivity to CsA parallel those of the human cyclophilins.
Zydowsky
L D
LD
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Ho
S I
SI
Baker
C H
CH
McIntyre
K
K
Walsh
C T
CT
D000591:Amino Acid Isomerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D016572:Cyclosporine / Q000378:metabolism* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D019696:Peptidylprolyl Isomerase; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D016572:Cyclosporine; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase