Expression of a cDNA for hippocalcin from rat brain

ABSTRACT

A hippocalcin cDNA from rat brain cDNA library was amplified by polymerase chain reation(PCR) and cloned using TA Cloning technique. For this PCR cloning, 29mer and 28mer oligonucleotide primers containing BamHl and EcoRl sites at the 5' end and 3' end, respectively were used. The nucleotide sequence of hippocalcin cDNA c1one was determined, and the complete amino acid sequence was deduced. Recombinant clone contained a cDNA insert of 610 base pairs with 582 nucleotides of open reading frame including the temination codon, 23 nucleotide of 5'-untranslated region, and 5nucleotides of 3'-nutran,slated region. The open reading frame encoded a polypepetid comprising 193 amino acids with molecular weight of 22kDa. The cDNA insert was subcloned into pVLI393 Baculovirus transfer vector. The recombinant hippocalcin was expressed in insect cell(Sf9 cell) using expression vector pVL1393. The hippocalcin expressed was purified as a single band on polyacrylamide gel electrophoresis following hydrophobic phenyl HPLC and TSKgel G3000SW gel filtration HPLC. Molecular size of rat brain hippocalcin protein expressed in this system was estimated to be 22kDa. Myristoylated hippocalcin migrated faster than nonmyristoyated form on SDS-polyacrylamide gel. Less than 10% of total hippocalcin expressed was myristoylated in this baculovirus expression system.