Baculovirus-mediated expression of recombinant rat phosphatidylcholine transfer protein.
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 approximately 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
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Marion Bessin Liver Research Center, Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Cohen
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D000595:Amino Acid Sequence; D000727:Androgen-Binding Protein; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D049294:Phospholipid Transfer Proteins; D060789:Prostatein; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification; D060765:Secretoglobins; D018411:Spodoptera / Q000378:metabolism; D014598:Uteroglobin; D060788:Mammaglobin B
D000727:Androgen-Binding Protein; D002352:Carrier Proteins; D018076:DNA, Complementary; D010446:Peptide Fragments; D049294:Phospholipid Transfer Proteins; D060789:Prostatein; D011994:Recombinant Proteins; D060765:Secretoglobins; D014598:Uteroglobin; C511484:Scgb1d2 protein, rat; C511483:Scgb1d4 protein, rat; C556468:Scgb2a1 protein, rat; D060788:Mammaglobin B