Tissue Factor


IUCr Abstract


Crystallization of 5G9-TF complex

26. Ruf, W., Stura, E.A., LaPolla, R.J., Syed, R., Edgington, T.S., Wilson, I.A. (1992) Purification, Sequence and Crystallization of an anti-Tissue Factor Fab and its use for the crystallization of Tissue Factor. Journal of Crystal Growth 122, 253-264.

Abstract: The crystallization of proteins as Fab-antigen complexes may offer several advantages over the crystallization of the uncomplexed proteins. Such advantages arise from the number of monoclonal antibodies that can be generated against a given protein. From each of these, Fab fragments can be generated providing new opportunities for crystallization of the protein as a complex. Further advantages may be realized by the possibility that protein-Fab structures can be solved by molecular replacement rather than the classical methodology of multiple isomorphous replacement (MIR). With this strategy, less protein may be required for the ligand-Fab structure solution compared to the MIR approach for solving the ligand alone. Here we report the screening and identification of a suitable Fab in the crystallization of the soluble extracellular domain of tissue factor, the receptor responsible for the cellular initiation of the coagulation protease cascade. From six specific Fabs, three have been identified that crystallize readily as free Fabs.The refinement of the purification procedure for one of these Fabs (TF8-5G9) was required to provide material which reliably formed complex crystals with the tissue factor extracellular domain. A further improvement in the quality of the complex crystals was achieved by enzymatic removal of sialic acid from tissue factor resulting in a significant reduction of its the charge heterogeneity. This study demonstrates the success of using Fabs in crystallization of a biologically important glycoprotein.

Structure paper in preparation by Mingdong Huang.


Crystallization of TF-fVIIa-5L15 complex

53. Stura, E. A., Ruf, W., Wilson I. A. (1996) Crystallization and preliminary crystallographic data for a ternary complex between tissue factor, factor VIIa and a BPTI-derived inhibitor. J. Cryst. Growth (In Press).

Abstract: The binding of tissue factor (TF) with the serine protease coagulation factor VIIa (VIIa) is the initial trigger for activation of the coagulation cascades. In complex with TF, VIIa has greatly enhanced proteolytic function in the activation of the natural substrate factors X and IX. A structural understanding of the interaction of VIIa with TF and how VIIa can be inhibited may help in the development of an anticoagulant strategy. Here we report the screening and identification of crystallization conditions to produce diffraction quality crystals of the complex between TF-VIIa and a potent inhibitor (5L15) derived from bovine pancreatic trypsin inhibitor (BPTI) by random mutagenesis and selection by phage display. The crystals were obtained from the soluble extracellular domain of tissue factor, expressed in Escherichia coli as a fusion protein, VIIa expressed in mammalian cells and recombinant 5L15. Because only 1.5 mg of complex were available for this work, a reverse screening based strategy was used in the search and optimization of the crystallization conditions. Two different crystal forms were obtained from polyethylene glycol 4,000 and monomethyl polyethylene glycol 2,000 with cacodylate buffer at pH 6.5 in the presence of sodium and calcium ions. The addition of magnesium, zinc and other divalent metals has profound effects on the crystallization. Both crystal forms are trigonal with cell parameters a = b = 129.3 Å, c=100.8 Å and a = b = 67.4 Å, c=317.1 Å diffracting to 6 Å and 3 Å resolution with one molecule in the asymmetric unit. Complete data sets have been collected from each of these to the resolution to which the crystals diffract.