|Li, Xin liang|
Submitted to: Protein and Peptide Letters
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/30/2006
Publication Date: 1/1/2006
Citation: Chen, H., Li, X., Xu, H., Ljungdahl, L.G., Cerniglia, C.E. 2006. High level expression and characterization of the cyclophilin B gene from the anaerobic fungus Orpinomyces sp. strain PC-2. Protein and Peptide Letters. 13:727-732. Interpretive Summary: Cyclophilins are widespread proteins found from bacteria to human. They have many functions including protein folding and serve as receptors for cyclosporine A, an important immunosuppressive drug commonly used to treat organ transplant patients. During the course of studies on biomass converting enzymes, we accidentally discovered a gene from Orpinomyces PC-2, an anaerobic fungus isolated from the rumen of a cow, coding for a cyclophilin that is very similar to the cyclophilin of cows. The data suggest that the gene in the fungus might have come from a cow during the history of evolution. The present study deals with the high yield production of the Orpinomyces cyclophilin in the bacterium Escherichia coli and further characterization of purified recombinant protein. The procedure developed will help us to prepare sufficient amounts of the protein for the elucidation of structure/function insights of such an important molecule.
Technical Abstract: Cyclophilins are an evolutionarily conserved family of peptidyl-prolyl cis-trans isomerases (PPIases). A cyclophilin B (CyPB) gene from anaerobic fungus Orpinomyces sp. strain PC-2 was cloned and overexpressed in Escherichia coli. It was expressed as amino terminal 6 x His-tagged recombinant protein to facilitate purification. Highly purified protein (26.5 kDa) was isolated by two chromatography steps for biochemical studies of the enzyme. The recombinant CyPB displayed PPIase activity with a kcat/Km, 8.9 x 10**6 M**-1 s**-1 at 10 deg C and pH 7.8 which is inhibited by cyclosporine A (CsA) with an IC50, 23.5 nM and similar to those of native protein and other cyclophilin B enzymes from animals. Genomic DNA analysis of cypb revealed it was present as a single copy in Orpinomyces PC-2 and contained two introns indicating it has a eukaryotic origin and is one of the most heavily interrupted genes by intron sequences found in anaerobic fungi.