Abstract

HERNANDEZ-CARVAJAL, Erick; ARCE-SOLANO, Silvia; MENA-AGUILAR, Didier  and  FUENTES-PRIOR, Pablo. Heterologous production and biochemical characterization of human pro-coagulation Factor VIII for crystallization screening of protein macromolecules. Tecnología en Marcha [online]. 2016, vol.29, n.4, pp.78-91. ISSN 0379-3982.  http://dx.doi.org/10.18845/tm.v29i4.3039.

In spite of their medical relevance and economic impact on public health service systems, cardiovascular diseases, among them myocardial and cerebral infarctions, remain the main cause of morbidity and mortality in developed and developing countries. One of the main reasons for this situation is the incomplete structural and functional knowledge of the processes that ultimately lead to blood clot formation. Thrombin plays a key role in these processes, but its molecular interactions with other clotting factors are still only poorly understood, in particular those with critically important substrates such as factor VIII and the thrombin receptor on platelets, PAR1. Given the importance of these clotting factors, we have produced and characterized factor VIII fragments (FVIII) to search for crystallization conditions of FVIII·Thrombin complexes. To achieve our aims, we (1) overexpressed the acidic inter-domain linkers of human factor VIII (FVIIIa1, FVIIIa2 and FVIIIa3), as heterologous proteins; (2) purified and characterized these recombinant forms, (3) formed the binary protein complexes between thrombin and these recombinant fragments, and (4) started crystallization trials of these complexes. The FVIII production, and in particular, the identification of conditions in which crystals of appropriate size and quality for structure determination grow, are a “bottleneck” in structure-and-function projects. Therefore, the optimization of these processes will allow us to obtain well-ordered protein crystals for X-ray diffraction studies.

Keywords : Cardiovascular disease; thrombin; FVIII; protein crystallization; X-ray diffraction.

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