Cloning and overexpression of an important functional gene ATP6V1F encoding a component of vacuolar ATPase from the Giant Panda (Ailuropoda melanoleuca).

ABSTRACT

ATP6V1F encodes a component of vacuolar ATPase mediating acidification. The cDNA and the genomic sequences of ATP6V1F were cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using reverse transcription polymerase chain reaction and touchdown-polymerase chain reaction, respectively. The cDNA fragment cloned is 364 bp in size, containing an open reading frame of 360 bp encoding 119 amino acids. Alignment analysis indicated that both ORF and the deduced amino acid sequence are highly conserved. The length of the genomic sequence of the Giant Panda is 2225 bp, including two exons and one intron. Topology prediction showed that there is one protein kinase C phosphorylation site, two Casein kinase II phosphorylation sites, and one N-myristoylation site in the ATP6V1F protein. The ATP6V1F gene was overexpressed in Escherichia coli indicating that ATP6V1F fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 17 kDa polypeptide, which was according with the predicted protein and also could be used to purify the protein and study its function.