Abstract
Phosphatidylcholine transfer protein catalyzes intermembrane transfer of phosphatidylcholines exclusive of all other phospholipid classes. Although postulated to participate in phosphatidylcholine biosynthesis and biliary trafficking in liver, the molecular basis underlying the substrate specificity of phosphatidylcholine transfer protein remains to be elucidated. Having demonstrated the inability of Escherichia coli to express recombinant phosphatidylcholine transfer protein, we infected Spodoptera frugiperda (Sf9) cells with recombinant baculovirus. When assayed in vitro, cytosol of recombinant but not control infected cells demonstrated high levels of intermembrane phosphatidylcholine transfer activity and no transfer activity for phosphatidylethanolamine. A two-step purification protocol in which 10 mg of cytosolic protein was subjected to anion exchange chromatography followed by hydroxylapatite chromatography yielded 0.1 mg active protein which was >92% pure. The identity of purified protein was confirmed by matrix-assisted laser desorption–ionization mass spectrometry and by amino acid sequencing. Based on the recovery of 30% of PC transfer activity after purification, we estimate that recombinant rat phosphatidylcholine transfer protein accounted for ∼3–6% of cytosolic protein mass of infected cells. ▪ These results demonstrate the utility of baculovirus for expressing recombinant phosphatidylcholine transfer protein and should facilitate studies designed to elucidate the structural biology and physiological functions of this uniquely specific phospholipid transfer protein.—Feng, L., and D. E. Cohen. Baculovirus-mediated expression of recombinant rat phosphatidylcholine transfer protein.
Highlights
Phosphatidylcholine transfer protein catalyzes intermembrane transfer of phosphatidylcholines exclusive of all other phospholipid classes
Whereas E. coli has been used to express recombinant sterol carrier protein 2 [10,11,12] and phosphatidylinositol transfer protein [13, 14], which are cytosolic proteins capable of promoting intermembrane transfer of PC in addition to other phospholipids, we have found this prokaryotic expression system to be inadequate for expression of recombinant phosphatidylcholine transfer protein (PC-TP).2
To ensure that the absence of PE transfer activity reflected specificity of the recombinant protein and not sequestration of [14C] PE within the inner hemileaflet where it would be inaccessible to the transfer protein [25], these results were verified using small unilamellar vesicles (SUV) composed of an equimolar mixture of PC and PE [26]
Summary
Phosphatidylcholine transfer protein catalyzes intermembrane transfer of phosphatidylcholines exclusive of all other phospholipid classes. Based on the recovery of 30% of PC transfer activity after purification, we estimate that recombinant rat phosphatidylcholine transfer protein accounted for ϳ3–6% of cytosolic protein mass of infected cells These results demonstrate the utility of baculovirus for expressing recombinant phosphatidylcholine transfer protein and should facilitate studies designed to elucidate the structural biology and physiological functions of this uniquely specific phospholipid transfer protein.—Feng, L., and D. Recent cDNA cloning of PC-TP from rat and bovine liver [5,6,7,8] has demonstrated these homologues to be highly similar (80% and 76% nucleotide and amino acid identities, respectively), but to be unrelated to other cytosolic lipid transfer proteins [9] These collective observations notwithstanding, the molecular basis underlying the unique specificity of PC-TP for PC as well as its function(s) in vivo have remained largely unresolved.
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call