Molecular cloning and expression analysis of phospholipase Cδ from mud loach, Misgurnus mizolepis

A gene encoding phosphoinositide-specific phospholipase C (PLC), designated ML-PLCdelta, was cloned from mud loach (Misgurnus mizolepis) liver. A complete cDNA encoding ML-PLCdelta was isolated by screening the cDNA library of mud loach liver and using the 5'rapid amplification of cDNA ends (RACE) method. The full-length ML-PLCdelta gene contains an open reading frame of 2325 base pairs encoding a 774 amino acid protein with a molecular mass of 88,072 Da; this corresponds to the size of the protein expressed in Escherichia coli BL21 (DE3) using pET28a vector. It contains all of the characteristic domains found in mammalian PLCdelta isozymes (PH domain, EFhands, X-Y catalytic region, and a C2 domain). A homology search revealed that ML-PLCdelta shares relatively high sequence identity with mammalian PLCdelta1 (51-52%) and catfish PLCdelta (64%). The recombinant ML-PLCdelta protein expressed as a hisfidine-tagged fusion protein in E. coli was purified to apparent homogeneity by Ni2+-NTA affinity chromatography. The recombinant ML-PLCdelta showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bis-phosphate (PIP2) and its activity was Ca2+ -dependent, which was similar to mammalian PLCdelta isozymes. (C) 2004 Elsevier Inc. All rights reserved.