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Development of Efficient Eukaryotic and Bacterial Expression Systems for Functional Studies of Recombinant Proteins of Biomedical Interest”

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Development of Efficient Eukaryotic and Bacterial Expression Systems for Functional Studies of Recombinant Proteins of Biomedical Interest” ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi doctoral i la seva utilització ha de respectar els drets de la persona autora. Pot ser utilitzada per a consulta o estudi personal, així com en activitats o materials dʼinvestigació i docència en els termes establerts a lʼart. 32 del Text Refós de la Llei de Propietat Intel·lectual (RDL 1/1996). Per altres utilitzacions es requereix lʼautorització prèvia i expressa de la persona autora. En qualsevol cas, en la utilització dels seus continguts caldrà indicar de forma clara el nom i cognoms de la persona autora i el títol de la tesi doctoral. No sʼautoritza la seva reproducció o altres formes dʼexplotació efectuades amb finalitats de lucre ni la seva comunicació pública des dʼun lloc aliè al servei TDX. Tampoc sʼautoritza la presentació del seu contingut en una finestra o marc aliè a TDX (framing). Aquesta reserva de drets afecta tant als continguts de la tesi com als seus resums i índexs. ADVERTENCIA. El acceso a los contenidos de esta tesis doctoral y su utilización debe respetar los derechos de la persona autora. Puede ser utilizada para consulta o estudio personal, así como en actividades o materiales de investigación y docencia en los términos establecidos en el art. 32 del Texto Refundido de la Ley de Propiedad Intelectual (RDL 1/1996). Para otros usos se requiere la autorización previa y expresa de la persona autora. En cualquier caso, en la utilización de sus contenidos se deberá indicar de forma clara el nombre y apellidos de la persona autora y el título de la tesis doctoral. No se autoriza su reproducción u otras formas de explotación efectuadas con fines lucrativos ni su comunicación pública desde un sitio ajeno al servicio TDR. Tampoco se autoriza la presentación de su contenido en una ventana o marco ajeno a TDR (framing). Esta reserva de derechos afecta tanto al contenido de la tesis como a sus resúmenes e índices. WARNING. The access to the contents of this doctoral thesis and its use must respect the rights of the author. It can be used for reference or private study, as well as research and learning activities or materials in the terms established by the 32nd article of the Spanish Consolidated Copyright Act (RDL 1/1996). Express and previous authorization of the author is required for any other uses. In any case, when using its content, full name of the author and title of the thesis must be clearly indicated. Reproduction or other forms of for profit use or public communication from outside TDX service is not allowed. Presentation of its content in a window or frame external to TDX (framing) is not authorized either. These rights affect both the content of the thesis and its abstracts and indexes. UNIVERSITAT AUTÒNOMA DE BARCELONA Departamento de Bioquímica y Biología Molecular Programa de Doctorado en Bioquímica, Biología Molecular y Biomedicina CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS INSTITUTO DE BIOLOGÍA MOLECULAR DE BARCELONA Structural Biology Unit Proteolysis Lab Development of efficient eukaryotic and bacterial expression systems for functional studies of recombinant proteins of biomedical interest Memoria presentada por Laura Mariño Puertas para optar al título de doctor por la Universitat Autònoma de Barcelona Doctorando: Laura Mariño Puertas Supervisores: Prof.F.Xavier Gomis-Rüth Dr.Theodoros Goulas Dr.Iñaki de Diego Martínez Tutor: Dr.Sandra Villegas Barcelona, Diciembre 2019 A todos los que siempre confiaron en mí, « Wanderer above the Sea of Fog » (1817-18), C. D. Friedrich “Life is not easy for any of us. But what of that? We must have perseverance and above all confidence in ourselves. We must believe that we are gifted for something and that this thing must be attained.” “La vida no es fácil, para ninguno de nosotros. Pero… ¡qué importa! Hay que perseverar y, sobre todo, tener confianza en uno mismo. Hay que sentirse dotado para realizar alguna cosa y que esa cosa hay que alcanzarla, cueste lo que cueste.” Marie Curie ACKNOWLEDGEMENTS No es fácil hacer una carta de agradecimientos, nada fácil, porque si soy sincera, ¡tengo mucho que agradecer! En primer lugar, me gustaría dar las gracias de corazón a Xavi, por traerme de Inglaterra y hacerme un hueco en su laboratorio, y por no haberme dicho nunca que no a ninguna de mis ideas, ¡¡por muy cara que fuera!! (incubador de Expi, estancia en Suiza, curso EMBO en Oxford, curso de cristalografía en Granada…). ¡¡Me ha permitido crecer enormemente en el terreno profesional y aún más importante, a creer en mis ideas y en mí misma!! GRACIAS. Tampoco puedo dejar en el tintero a mis supervisores de tesis, a Iñaki y Theo. Iñaki me ha enseñado a tomarme menos en serio y con él viví mi primer sincrotrón (en Grenoble, Francia) y eso nunca se olvida (aunque sólo sea por el miedo que dan las instalaciones.). De Theo he aprendido perseverancia, a ser un poco más paciente y, ¡mucho más práctica! Ambos me han hecho mejor científica, ¡¡así que estaré siempre en duda con ellos!! A todos los compis actuales y pasados del lab, que sois mucho más que compañeros, sois amigos, compañeros de juegos y ha sido un placer compartir estos cinco años con vosotros… ¡y que estoy segura de que serán muchos más! Tampoco me quiero olvidar de todos los compañeros y personal del Parc Cientific, que habéis conseguido que nunca me sintiera sola allí, ¡¡ni siquiera a horas intempestivas!! Debo también agradecer a mis colaboradores, que me han prestado desinteresadamente, vectores, protocolos y resuelto dudas. Gracias, vosotros le dais sentido a la ciencia, porque si no fuese compartido el conocimiento, ¡no sería ciencia! También le quiero agradecer a Alex su comprensión, ¡¡porque no es fácil tener una novia chiflada, que tiene ideas científicas cepillándose los dientes y que su idea de domingo sea ir a pasar las células!! Además, él ha sido parte fundamental en la corrección de la tesis, leyéndosela entera y dándome su feedback en inglés. ¡¡Sin él, esta tesis no sería ni parecida!! Y, por último, quiero agradecer a mi familia por su apoyo incondicional. Porque no tiene que ser nada fácil tener una hija a la que le encanta estar lejos de casa, pasando aventuras y viviendo en la distancia sus logros y penas…¡¡pero sin ese apoyo en la distancia, jamás habría llegado hasta dónde lo he hecho!! Y por favor, si me olvido de alguien, no me lo tengáis en cuenta, todos los que habéis formado parte de estos cinco años, para bien y para mal, ¡habéis sido importantes! I II DECLARATION OF AUTHORSHIP I, Laura Mariño Puertas, declare that this thesis and the work presented in it are my own and has been generated by me as the result of my own original research: “Development of efficient eukaryotic and bacterial expression systems for functional studies of recombinant proteins of biomedical interest” I confirm that: - This work was done wholly while in candidature for a research degree at this University; - Where I have consulted the published work of others, this is always clearly attributed; - Where I have quoted from the work of others, the source is always given. With the exception of such quotations, this thesis is entirely my own work; - I have acknowledged all main sources of help; - Where the thesis is based on work done by myself jointly with others, I have made clear exactly what was done by others and what I have contributed myself; There are parts of this work that have been published as scientific publications: • Goulas T, Garcia-Ferrer I, Marrero A, Marino-Puertas L, Duquerroy S, Gomis-Rüth FX. (2017). Structural and functional insight into pan-endopeptidase inhibition by α2-macroglobulins. Biological Chemistry, 398(9):975-994. doi: 10.1515/hsz-2016-0329. - Marino-Puertas L., Goulas T and Gomis-Rüth FX. (2017). Matrix metalloproteinases outside vertebrates. Biochimica Biophysica Acta Molecular Cell Research, 1864(11 Pt A):2026-2035. doi: 10.1016/j.bbamcr.2017.04.003. Epub 2017 Apr 7. Review. - Del Amo-Maestro, L., Marino-Puertas, L., Goulas, T. & Gomis-Rüth, F. X. (2019). Recombinant production, purification, crystallization, and structure analysis of human transforming growth factor β2 in a new conformation. Scientific reports, 9(1), 8660. - Marino-Puertas, L., Del Amo-Maestro, L., Taulés, M., Gomis-Rüth, F. X., & Goulas, T. (2019). Recombinant production of human α2-macroglobulin variants and interaction studies with recombinant G-related α2-macroglobulin binding protein and latent transforming growth factor-β2. Scientific reports, 9(1), 9186. Signed: Date: III IV ABSTRACT In this thesis, efforts have been made to develop a system for heterologous expression of human glycoproteins with high yield, purity and homogeneity, as well as the necessary flexibility to engineer the protein at will. We established two eukaryotic expression systems, based in Drosophila Schneider 2 cells (inducible expression of stable cell lines by addition of Cu2SO4 and transient expression with polyethylenimine (PEI) transfection) and in mammalian cells (transient expression on Expi293F cells with PEI transfection). Proteolysis is a fundamental part of the protein turnover in living organisms and its imbalance may lead to diseases, causing inflammation, neurodegenerative diseases, cancer or cell death among other disorders. Thus, mechanisms that regulate peptidase activity must be exquisitely regulated to ensure proper proteolytic activity at the right time and place. The human subtilisin kexin-isozyme 1 (hSKI1) is the serine protease which catalyzes the first step in the proteolytic activation of the sterol regulatory element binding proteins (SREBPs), key regulator of the intracellular lipid metabolism. This protease is a transmembrane protein localized in endoplasmic reticulum and Golgi apparatus and its maturation is a very complex process. We achieved to express it soluble in the insect and mammalian expression systems and laid the foundations for future biochemical and structural studies.
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