Molecular cloning and expression of a human brain inositol 1,4,5-trisphosphate 3-kinase

Abstract

A human hippocampus cDNA library was screened by hybridization with a rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase cDNA. Sequencing of three overlapping clones identified a 1383 bp open reading frame encoding a 461 amino acid protein with a calculated molecular weight of 50988. The coding amino acid sequence showed an overall 93% similarity with the sequence of rat brain InsP3 3-kinase. The cDNA insert of one isolated partial clone (i.e. hh 26) was in frame with the beta galactosidase fragment fused to it as a Bluescript plasmid; it displayed InsP3 3-kinase activity when expressed in Escherichia coli (E. coli). Biochemical characterization of human brain InsP3 3-kinase by SDS polyacrylamide gel electrophoresis and regeneration of enzyme activity reveals three active fractions with apparent Mr of 58,000-64,000, 45,000-50,000 and 37,000-39,000.