Structure and expression of cytosolic cyclophilin/peptidyl-prolyl cis-trans isomerase of higher plants and production of active tomato cyclophilin in Escherichia coli.
cDNA clones encoding proteins of approximately 18 kDa in which 83% of the amino acids are conserved relative to the published sequences of mammalian cyclophilin/rotamase (CyP) have been isolated from tomato, maize, and Brassica napus. In correspondence with the mammalian genes, but in contrast with the Neurospora gene and one yeast CyP gene, the plant CyP genes encode only mature proteins lacking transit peptides. RNA blot analyses demonstrate that CyP genes are expressed in all plant organs tested. Southern blots of genomic DNA indicate that there are small families (two to eight members) of CyP-related genes in maize and B. napus. A vector was constructed for expression of the tomato cDNA in E. coli. SDS/polyacrylamide gels show that extracts of appropriately induced cells harboring this vector contain nearly 40% of the protein as a single approximately 18-kDa band. While the majority of this protein is sequestered in insoluble inclusion bodies, the soluble extracts have higher levels of peptidyl-prolyl cis-trans isomerase (rotamase) activity than extracts of wild-type cells. This additional activity is sensitive to inhibition by the cyclic undecapeptide cyclosporin A.
Gasser
C S
CS
Department of Biochemistry and Biophysics, University of California, Davis 95616.
Gunning
D A
DA
Budelier
K A
KA
Brown
S M
SM
D000591:Amino Acid Isomerases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D019696:Peptidylprolyl Isomerase; D010944:Plants / Q000201:enzymology / Q000235:genetics*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D002352:Carrier Proteins; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D012313:RNA; D004247:DNA; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase