Expression, purification, and spectroscopic characterization of human thromboxane synthase.
Thromboxane A2 (TXA2) is a potent inducer of vasoconstriction and platelet aggregation. Large scale expression of TXA2 synthase (TXAS) is very useful for studies of the reaction mechanism, structural/functional relationships, and drug interactions. We report here a heterologous system for overexpression of human TXAS. The TXAS cDNA was modified by replacing the sequence encoding the first 28 amino acid residues with a CYP17 amino-terminal sequence and by adding a polyhistidine tag sequence prior to the stop codon; the cDNA was inserted into the pCW vector and co-expressed with chaperonins groES and groEL in Escherichia coli. The resulting recombinant protein was purified to electrophoretic homogeneity by affinity, ion exchange, and hydrophobic chromatography. UV-visible absorbance (UV-Vis), magnetic circular dichroism (MCD), and electron paramagnetic resonance (EPR) spectra indicate that TXAS has a typical low spin cytochrome P450 heme with an oxygen-based distal ligand. The UV-Vis and EPR spectra of recombinant TXAS were essentially identical to those of TXAS isolated from human platelets, except that a more homogenous EPR spectrum was observed for the recombinant TXAS. The recombinant protein had a heme:protein molar ratio of 0.7:1 and a specific activity of 12 micromol of TXA2/min/mg of protein at 23 degreesC. Furthermore, it catalyzed formation of TXA2, 12-hydroxy-5,8,10-heptadecatrienoic acid, and malondialdehyde in a molar ratio of 0.94:1.0:0.93. Spectral binding titrations showed that bulky heme ligands such as clotrimazole bound strongly to TXAS (Kd approximately 0.5 microM), indicating ample space at the distal face of the heme iron. Analysis of MCD and EPR spectra showed that TXAS was a typical low spin hemoprotein with a proximal thiolate ligand and had a very hydrophobic distal ligand binding domain.
Hsu
P Y
PY
Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center, Houston, Texas 77030, USA.
Tsai
A L
AL
Kulmacz
R J
RJ
Wang
L H
LH
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D006418:Heme / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015203:Reproducibility of Results; D013057:Spectrum Analysis; D013930:Thromboxane-A Synthase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D008024:Ligands; D011994:Recombinant Proteins; D006418:Heme; D013930:Thromboxane-A Synthase