OVEREXPRESSION, PURIFICATION AND CHARACTERIZATION OF INOSITOL 1,4,5-TRISPHOSPHATE 3-KINASE FROM RAT-BRAIN

Abstract

The entire gene encoding D-myo-inositol 1,4,5-trisphosphate 3-kinase (IP3K) of rat brain was overexpressed using a T7 promoter in an expression vector pT7-7. When induced by isopropyl-beta-D-thiogalactoside, E. coli BL21(DE3) transformed with pT7-7/IP3K expressed IP3K over 130 fold compared with rat brain. IP3K was purified to homogeneity by a simple and rapid purification procedure utilizing a calmodulin-agarose column as a major chromatographic step. The purified IP3K exhibited a polypeptide (M(r) = 50000) on sodium dodecylsulfate-polyacrylamide gel electrophoresis which shows specific cross-reactivity towards monoclonal antibodies raised against IP3K from rat brain. The purified IP3K has a specific activity of 20 mu mole/min/mg protein in the absence of calmodulin, with 1.5 fold activation of the activity by Ca2+/calmodulin.