<?xml version="1.0" encoding="ISO-8859-1"?><article xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance">
<front>
<journal-meta>
<journal-id>0379-3982</journal-id>
<journal-title><![CDATA[Revista Tecnología en Marcha]]></journal-title>
<abbrev-journal-title><![CDATA[Tecnología en Marcha]]></abbrev-journal-title>
<issn>0379-3982</issn>
<publisher>
<publisher-name><![CDATA[Instituto Tecnológico de Costa Rica]]></publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id>S0379-39822019000400039</article-id>
<article-id pub-id-type="doi">10.18845/tm.v32i4.4789</article-id>
<title-group>
<article-title xml:lang="es"><![CDATA[Mutagénesis dirigida del conector interdominio ácido FVIIIa3 del factor VIII de la coagulación como estrategia para favorecer la cristalización de sus complejos con la trombina]]></article-title>
<article-title xml:lang="en"><![CDATA[Site-directed mutagenesis of the acidic interdomain linker FVIIIa3 of the coagulation factor VIII as a strategy to favor crystallization of its complexes with thrombin]]></article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname><![CDATA[Rojas-Murcia]]></surname>
<given-names><![CDATA[Jeremy]]></given-names>
</name>
<xref ref-type="aff" rid="Aff"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname><![CDATA[Arce-Solano]]></surname>
<given-names><![CDATA[Silvia]]></given-names>
</name>
<xref ref-type="aff" rid="Aff"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname><![CDATA[Fuentes-Prior]]></surname>
<given-names><![CDATA[Pablo]]></given-names>
</name>
<xref ref-type="aff" rid="Aff"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname><![CDATA[Hernández-Carvajal]]></surname>
<given-names><![CDATA[Erick]]></given-names>
</name>
<xref ref-type="aff" rid="Aff"/>
</contrib>
</contrib-group>
<aff id="Af1">
<institution><![CDATA[,Instituto Tecnológico de Costa Rica Centro de Investigación en Biotecnología ]]></institution>
<addr-line><![CDATA[ ]]></addr-line>
<country>Costa Rica</country>
</aff>
<aff id="Af2">
<institution><![CDATA[,Instituto Tecnológico de Costa Rica Ing. Licenciada en Biotecnología Centro de Investiga-ción en Biotecnología]]></institution>
<addr-line><![CDATA[ ]]></addr-line>
<country>Costa Rica</country>
</aff>
<aff id="Af3">
<institution><![CDATA[,Institut d&#8217;Investigació Biomèdica (IIB) Doctor en Bioquímica Sant Pau, Hospital de la Santa Creu i Sant Pau]]></institution>
<addr-line><![CDATA[ Barcelona]]></addr-line>
<country>España</country>
</aff>
<aff id="Af4">
<institution><![CDATA[,Instituto Tecnológico de Costa Rica Centro de Investigación en Bio-tecnología ]]></institution>
<addr-line><![CDATA[ ]]></addr-line>
<country>Costa Rica</country>
</aff>
<pub-date pub-type="pub">
<day>00</day>
<month>12</month>
<year>2019</year>
</pub-date>
<pub-date pub-type="epub">
<day>00</day>
<month>12</month>
<year>2019</year>
</pub-date>
<volume>32</volume>
<numero>4</numero>
<fpage>39</fpage>
<lpage>50</lpage>
<copyright-statement/>
<copyright-year/>
<self-uri xlink:href="http://www.scielo.sa.cr/scielo.php?script=sci_arttext&amp;pid=S0379-39822019000400039&amp;lng=en&amp;nrm=iso"></self-uri><self-uri xlink:href="http://www.scielo.sa.cr/scielo.php?script=sci_abstract&amp;pid=S0379-39822019000400039&amp;lng=en&amp;nrm=iso"></self-uri><self-uri xlink:href="http://www.scielo.sa.cr/scielo.php?script=sci_pdf&amp;pid=S0379-39822019000400039&amp;lng=en&amp;nrm=iso"></self-uri><abstract abstract-type="short" xml:lang="es"><p><![CDATA[Resumen Las enfermedades cardiovasculares representan una de las principales causas de morbilidad y mortalidad a nivel mundial, lo que se traduce en un fuerte impacto económico en el sector salud. El factor VIII (FVIII) es un cofactor esencial en los procesos hemostáticos que participa en la formación del coágulo ante alguna señal de daño vascular. La proteasa trombina es el principal activador fisiológico del FVIII, pero los mecanismos moleculares de reconocimiento del FVIII por la trombina y la formación de los complejos transitorios Michaelis-Menten correspondientes aún no se han descrito en detalle. La cristalografía de rayos X es una técnica que permite resolver la estructura 3D de complejos proteicos, pero requiere la cristalización previa de la muestra a estudiar. Con el objetivo de favorecer la formación de cristales del FVIIIa3 humano (residuos Glu1649-Arg1689) acomplejado con la trombina, se realizó mutagénesis dirigida para generar los doble-mutantes FVIIIa3 (R1689Q, G1690P) y FVIIIa3 (R1689G, G1690P). Estos fragmentos fueron sobreexpresados de forma heteróloga y purificados, obteniendo rendimientos de ~1.5 mg por cada litro de cultivo bacteriano. Esto permitió generar los complejos proteicos FVIIIa3 (R1689Q, G1690P)&#8226;trombina y FVIIIa3 (R1689G, G1690P)&#8226;trombina en cantidades suficientes para explorar sus espacios de solubilidad de forma extensiva. En total, en cada caso se evaluaron entre 630 y 820 condiciones de cristalización distintas. A partir de esta búsqueda inicial se obtuvo algún tipo de precipitado cristalino en 22 condiciones, 10 de las cuales lograron ser optimizadas para obtener monocristales de alta calidad para posteriores ensayos de difracción con rayos X.]]></p></abstract>
<abstract abstract-type="short" xml:lang="en"><p><![CDATA[Abstract Cardiovascular diseases are one of the most important causes of morbidity and mortality worldwide, which translates into a strong economic impact in the health sector. Factor VIII (FVIII) is an essential cofactor within the hemostatic processes involved in blood clot formation upon vascular damage. The proteinase thrombin is the most important physiological activator of FVIII, but the molecular mechanisms underlying recognition of FVIII by thrombin and the formation of the corresponding, transient Michaelis-Menten complexes are still only poorly understood. X-ray crystallography allows the resolution of the 3D structure of protein complexes, but a prerequisite is the crystallization of the sample to be analyzed. To grow crystals of the human FVIIIa3 (residues Glu1649-Arg1689) complexed with thrombin, site-directed mutagenesis was performed to generate the double-mutants FVIIIa3 (R1689Q, G1690P) and FVIIIa3 (R1689G, G1690P). Both fragments were overexpressed as heterologous proteins and purified with yields of ~1.5 mg per liter of bacterial culture. Availability of these recombinant proteins allowed in turn generation of FVIIIa3 (R1689Q, G1690P)&#8226;thrombin and FVIIIa3 (R1689G, G1690P)&#8226;thrombin complexes to perform extensive screenings of their solubility space. Altogether, a total of 630 to 820 different crystallization conditions were evaluated for both complexes. From these initial screen, some sort of crystalline precipitate was obtained in 22 conditions, 10 of which could be optimized to grow high-quality single crystals for subsequent X-ray diffraction analysis.]]></p></abstract>
<kwd-group>
<kwd lng="es"><![CDATA[Enfermedades cardiovasculares]]></kwd>
<kwd lng="es"><![CDATA[trombina]]></kwd>
<kwd lng="es"><![CDATA[factor VIII]]></kwd>
<kwd lng="es"><![CDATA[mutagénesis dirigida]]></kwd>
<kwd lng="es"><![CDATA[cristalización de proteínas]]></kwd>
<kwd lng="en"><![CDATA[Cardiovascular diseases]]></kwd>
<kwd lng="en"><![CDATA[thrombin]]></kwd>
<kwd lng="en"><![CDATA[factor VIII]]></kwd>
<kwd lng="en"><![CDATA[site-directed mutagenesis]]></kwd>
<kwd lng="en"><![CDATA[protein crystallization]]></kwd>
</kwd-group>
</article-meta>
</front><back>
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