CHARACTERIZATION OF MOUSE PHOSPHATIDYLINOSITOL TRANSFER PROTEIN EXPRESSED IN ESCHERICHIA-COLI

The cDNA encoding mouse phosphatidylinositol transfer protein (PI-TP) was isolated by means of reverse transcriptase polymerase chain reacti on. The nucleotide sequence of this cDNA has a high similarity (98%) w ith that of rat PI-TP; the predicted amino acid sequence is 99.6% iden tical to that of rat PI-TP. The cDNA encoding mouse PI-TP was cloned i nto the expression vector pET3d and the Escherichia coli strain BL21(D E3) was transformed with the resulting plasmid. After induction of the bacteria with isopropyl-beta-D-thiogalactopyranoside, PI-TP was effic iently expressed in the E. coli strain. It was estimated that 5% of th e total soluble cell protein consisted of PI-TP. The recombinant mouse PI-TP was purified from the bacterial lysate in four steps: ammonium sulphate precipitation, anion-exchange chromatography, heparin-Sepharo se affinity and gel filtration chromatography. Fractionation on the he parin-Sepharose affinity column yielded two forms: PI-TP Hepa1 and Hep a2. These two proteins have the same molecular mass of 35 kDa, both co ntain a phosphatidylglycerol molecule and both are recognized by anti- PI-TP antibody. Both recombinant proteins have an isoelectric point of 5.4 as compared to 5.5 for bovine brain PI-TP. Sequence analysis of t he first 25 N-terminal amino acid residues showed that both forms are identical, except that PI-TP Hepa1 contains the initiator methionine w hich is lacking from PI-TP Hepa2. The two PI-TP forms have similar pho spholipid-binding and transfer activity, comparable to that of bovine brain PI-TP. Both forms and bovine brain PI-TP are phosphorylated equa lly well in a Ca2+/phospholipid-dependent way by protein kinase C.