High level expression and characterization of the cyclophilin B gene from the anaerobic fungus Orpinomyces sp. strain PC-2.
Cyclophilins are an evolutionarily conserved family of peptidyl-prolyl cis-trans isomerases (PPIases). A cyclophilin B (cypB) gene from the anaerobic fungus Orpinomyces sp. strain PC-2 was cloned and overexpressed in Escherichia coli. It was expressed as an amino-terminal 6 x His-tagged recombinant protein to facilitate purification. Highly purified protein (26.5 kDa) was isolated by two chromatographic steps involving affinity and gel filtration for biochemical studies of the enzyme. The recombinant CypB displayed PPIase activity with a k(cat)/K(m) of 8.9 x 10(6) M(-1) s(-1) at 10 degrees C and pH 7.8. It was inhibited by cyclosporin A (CsA) with an IC(50) of 23.5 nM, similar to those of the native protein and other cyclophilin B enzymes from animals. Genomic DNA analysis of cypB revealed that it was present as a single copy in Orpinomyces PC-2 and contained two introns, indicating it has a eukaryotic origin. It is one of the most heavily interrupted genes with intron sequences found in anaerobic fungi. The three-dimensional model of Orpinomyces PC-2 CypB was predicted with a homology modeling approach using the Swiss-Model Protein Modeling Server and three dimensional structure of human CypB as a template. The overall architecture of the CypB molecule is very similar to that of human CypB.
Chen
Huizhong
H
Division of Microbiology, National Center for Toxicological Research, U.S. FDA, 3900 NCTR Rd., Jefferson, AR 72079, USA. huizhong.chen@fda.hhs.gov
Li
Xin-Liang
XL
Xu
Haiyan
H
Ljungdahl
Lars G
LG
Cerniglia
Carl E
CE
D001483:Base Sequence; D003001:Cloning, Molecular; D021983:Cyclophilins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D020085:Neocallimastigales / Q000235:genetics*; D019696:Peptidylprolyl Isomerase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
C081786:cyclophilin B; D021983:Cyclophilins; D019696:Peptidylprolyl Isomerase