cDNA, genomic sequence cloning and overexpression of cytochrome c oxidase gene (COX6b1) from the Ailuropoda melanoleuca

Abstract

Cytochrome c oxidase (COX) is a component of the mitochondria respiratory chain. COX6b1 is one of the COX small subunits encoded by nuclear genes. In currently study, the cDNA and the genomic sequence of COX6b1 were successfully cloned from the Ailuropoda melanoleuca with the RT-PCR technology and Touchdown-PCR, respectively. The length of cDNA fragment cloned is 341 bp in size, containing an open reading frame (ORF) of 261 bp encoding 86 amino acids. The length of the genomic sequence is 4657 bp, which was found to possess 3 exons and 2 introns. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved with other six species studied. Topology prediction showed that there are one N-glycosylation site, one protein kinase C phosphorylation site, two casein kinase II phosphorylation sites, two N-myristoylation sites and one microbodies C-terminal targeting signal site in the COX6b1 protein of the A. melanoleuca. The COX6b1 gene was also overexpressed in Escherichia coli and the result indicated that COX6b1 fusion with the HIS-tagged gave rise to the accumulation of an expected 13.46 kDa polypeptide. The  expression product obtained could be used to purify the protein and study its function further.

Key words: Cytochrome c oxidase subunit Vib polypeptide 1 (COX6b1), clone, overexpression, Ailuropoda melanoleuca (Giant Panda), sequences analysis.