RESUMO
Lysosomal storage diseases (LSDs) are caused by accumulation of partially degraded substrates within the lysosome, as a result of a function loss of a lysosomal protein. Recombinant lysosomal proteins are usually produced in mammalian cells, based on their capacity to carry out post-translational modifications similar to those observed in human native proteins. However, during the last years, a growing number of studies have shown the possibility to produce active forms of lysosomal proteins in other expression systems, such as plants and microorganisms. In this paper, we review the production and characterization of human lysosomal proteins, deficient in several LSDs, which have been produced in microorganisms. For this purpose, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Ogataea minuta have been used as expression systems. The recombinant lysosomal proteins expressed in these hosts have shown similar substrate specificities, and temperature and pH stability profiles to those produced in mammalian cells. In addition, pre-clinical results have shown that recombinant lysosomal enzymes produced in microorganisms can be taken-up by cells and reduce the substrate accumulated within the lysosome. Recently, metabolic engineering in yeasts has allowed the production of lysosomal enzymes with tailored N-glycosylations, while progresses in E. coli N-glycosylations offer a potential platform to improve the production of these recombinant lysosomal enzymes. In summary, microorganisms represent convenient platform for the production of recombinant lysosomal proteins for biochemical and physicochemical characterization, as well as for the development of ERT for LSD.
Assuntos
Doenças por Armazenamento dos Lisossomos/tratamento farmacológico , Lisossomos/enzimologia , Proteínas/isolamento & purificação , Proteínas Recombinantes/biossíntese , Animais , Escherichia coli/metabolismo , Vetores Genéticos/metabolismo , Humanos , Plantas/genética , Proteínas/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Saccharomycetales/metabolismoRESUMO
Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency. Currently no effective therapies exist for MPS IVA. In this work, production of a recombinant GALNS enzyme (rGALNS) in Escherichia coli BL21 strain was studied. At shake scale, the effect of glucose concentration on microorganism growth, and microorganism culture and induction times on rGALNS production were evaluated. At bench scale, the effect of aeration and agitation on microorganism growth, and culture and induction times were evaluated. The highest enzyme activity levels at shake scale were observed in 12 h culture after 2-4 h induction. At bench scale the highest enzyme activity levels were observed after 2 h induction. rGALNS amounts in inclusion bodies fraction were up to 17-fold higher than those observed in the soluble fraction. However, the highest levels of active enzyme were found in the soluble fraction. Western blot analysis showed the presence of a 50-kDa band, in both soluble and inclusion bodies fractions. These results show for the first time the feasibility and potential of production of active rGALNS in a prokaryotic system for development of enzyme replacement therapy for MPS IVA disease.
Assuntos
Condroitina Sulfatases/biossíntese , Escherichia coli/metabolismo , Fermentação , Corpos de Inclusão/microbiologia , Western Blotting , Condroitina Sulfatases/genética , Clonagem Molecular , Meios de Cultura , Terapia de Reposição de Enzimas/métodos , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Mucopolissacaridose IV/terapia , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
En la búsqueda de alternativas para mejorar la expresión de la enzima Iduronato Sulfatasa (IDSh) en la levaduraPichia pastoris, se realizó una Revisión Sistemática de la Literatura con el fin de recopilar información quepermitiera relacionar los niveles de expresión de proteínas humanas recombinantes con la señal de secreción y las características propias de la molécula a expresar. Se hallaron 349 publicaciones de las cuales sólo 7 (2)porciento reportaron la expresión de proteínas que cumplían con los criterios de inclusión manejados en el estudio. Con la información obtenida en los 7 artículos se realizó una prueba de hipótesis tomando como muestras los datos recopilados y un análisis cualitativo de la información, con los cuales se evidenció que al reemplazar la señal de secreción nativa por el a-Factor como péptido líder se incrementa el nivel de expresión de proteínas humanas recombinantes en P. pastoris (p=0.053). Se encontró que la eliminación de la secuencia que codifica para el péptido nativo heterólogo en el ADNc de la proteína, es imprescindible para que el a-Factor pueda favorecer la secreción de proteínas heterólogas y por consiguiente incrementar el nivel de expresión. En el caso de la IDShr se halló que en la construcción GS115/pPIC9-IDS, aparecen dos secuencias de péptido señal al mismo tiempo, la nativa de la IDSh y la putativa proveniente de Saccharomyces cerevisiae; sin embargo, se han obtenidos expresiones hasta de ~30mmol/h mg de proteína, lo que deja la incógnita de un posible conflicto en el reconocimiento erróneo de una u otra señal de secreción, teniendo en cuenta el grado de hidrofobicidad de ambas.