Development of constructs for recombinant expression of human follicle-stimulating hormone receptor in rod cells of the zebrafish

• Autor(a) principal: Carrola, Joana Carolina Quintela
• Data de Publicação: 2009
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/1342
• topic: Proteínas G; Rodopsina; Proteínas G - Receptores acoplados; Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química

The follicle-stimulating hormone (FSH) is involved in the regulation of reproduction, by acting through a G protein-coupled receptor (GPCR) on the surface of target cells. Like most of the GPCRs, not much is known about the structure of the follicle-stimulating hormone receptor (FSHR). It is relatively difficult to purify the FSHR protein from gonadal tissues, due to its low abundance on its native cells, and therefore, to study its structure. For these reasons it is necessary to come up with a strategy that allows the production of large quantities of protein in order to use it in studies to obtain detailed structural information on full-length human FSHR. Via polymerase chain reactions, site-directed mutagenesis and other molecular biological methods, we were able to construct mutated receptors with different signal sequences and different tags, including the last fifteen amino acids of human rhodopsin which allow the receptor to be expressed on retinal rod cells of zebrafish. Mutations were designed in a manner that the receptor becomes inactive, i.e. incapable of signal transduction but still expressed at cell surface. The receptor constructs were first tested in human embryonic kidney cells (HEK 293-T) and their inserts will be used for transgenetic studies on zebrafish in the future.