Bacterial expression of functional membrane-bound thromboxane synthase having intact sequence and truncated N-terminal hydrophobic segment.
A full-length cDNA for human placental thromboxane synthase and a shortened cDNA lacking the sequence corresponding to the N-terminal 2-29 amino acids were expressed in Escherichia coli using a pCW expression vector. Both intact and truncated recombinant enzyme were found in the membrane fraction and were catalytically active. These results suggest that the N-terminal hydrophobic segment, a proposed membrane anchor for P-450 enzymes, is not solely responsible for attachment of thromboxane synthase to the membrane and is not required for the proper protein folding or the enzyme activity.
Xia
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Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington 40536, USA.
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005260:Female; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010920:Placenta / Q000201:enzymology*; D010957:Plasmids; D011247:Pregnancy; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D017384:Sequence Deletion; D013347:Subcellular Fractions / Q000201:enzymology; D013930:Thromboxane-A Synthase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D013930:Thromboxane-A Synthase