Molecular cloning and expression analysis of phospholipase Cdelta 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, EF-hands, 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 histidine-tagged fusion protein in E. coli was purified to apparent homogeneity by Ni(2+)-NTA affinity chromatography. The recombinant ML-PLCdelta showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bis-phosphate (PIP(2)) and its activity was Ca(2+)-dependent, which was similar to mammalian PLCdelta isozymes.
Kim
Moo Sang
MS
Department of Biotechnology, College of Fisheries Sciences, Pukyong National University, 599-1, Daeyean 3 Dong, Nam-gu, Busan, 608-737, South Korea.
Seo
Jung Soo
JS
Choi
Gang Eun
GE
Lim
Sang Uk
SU
Chung
Joon Ki
JK
Lee
Hyung Ho
HH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D003531:Cypriniformes / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D004926:Escherichia coli; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D054803:Phospholipase C delta; D010802:Phylogeny; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D019269:Phosphatidylinositol 4,5-Diphosphate; D011993:Recombinant Fusion Proteins; D010738:Type C Phospholipases; D054803:Phospholipase C delta; D002118:Calcium