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Overproduction of human M3 muscarinic acetylcholine receptor: An approach toward structural studies

Romero-Fernandez, Wilber ; Garriga, Pere ; Borroto-Escuela, Dasiel O.

Biotechnology progress, 2011-05, Vol.27 (3), p.838-845 [Periódico revisado por pares]

Hoboken: Wiley Subscription Services, Inc., A Wiley Company

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Título:
Overproduction of human M3 muscarinic acetylcholine receptor: An approach toward structural studies
Autor: Romero-Fernandez, Wilber ; Garriga, Pere ; Borroto-Escuela, Dasiel O.
Assuntos: Biological and medical sciences ; Biotechnology ; Ciències de la salut ; codon optimization ; epitope tags ; Fundamental and applied biological sciences. Psychology ; G proteins ; G-protein-coupled receptors ; Gene Expression ; Genes, Neoplasm - genetics ; Humans ; Medicin och hälsovetenskap ; Medicina ; Methods ; Molecular Probes ; muscarinic receptor ; Muscarinic receptors ; Neoplasm Proteins - biosynthesis ; Neurologia ; overexpression ; Proteïnes G ; Receptor, Muscarinic M3 - biosynthesis ; Receptor, Muscarinic M3 - chemistry ; Receptor, Muscarinic M3 - genetics ; Receptors ; Semliki Forest virus ; Transfection ; Up-Regulation ; Àrees temàtiques de la UPC
É parte de: Biotechnology progress, 2011-05, Vol.27 (3), p.838-845
Notas: istex:623D0D0C4A3A01504E088377D740C10F629C93C9
ArticleID:BTPR615
Spanish Ministry of Education and Science - No. FPU-AP-2004-6658; No. SAF 2005-08148-C04-02; No. SAF 2008-04943-C02-02
European Social Foundation and the Government of Catalonia - No. FI2004; No. BE-2006
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Descrição: Human M3 muscarinic acetylcholine receptor (M3R), present in both the central and the peripheral nervous system, is involved in several neurodegenerative and autoimmune diseases. Recently, M3R overexpression has been suggested to play a role in certain forms of cancer, showing promise as a new potential pharmacological target. However, the lack of structural information hampered to develop a new potent selective and potent antagonist. We describe here different strategies for overexpressing functional M3R on the perspective of future biophysical studies. To achieve this goal, four tagged M3R genes were engineered and codon optimized. Different heterologous expression systems, including mammalian cells and viral transfection, were employed to overexpress M3R. Although codon optimization resulted in only twofold to threefold increase of M3R expression, we found that epitope tagging of the synthetic M3R, especially with hemagglutinin and Flag epitope tags, could improve M3R expression levels. On the other hand, viral transfection led to a yield of 27 pmol/mg protein that is the highest level reported so far for this receptor subtype in mammalian cells. Taking together several of the strategies used can help increasing M3R expression, not only to start purification efforts but also for secondary structural analysis trial and functional analyses. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
Editor: Hoboken: Wiley Subscription Services, Inc., A Wiley Company
Idioma: Inglês

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Overproduction of human M3 muscarinic acetylcholine receptor: An approach toward structural studies

Romero-Fernandez, Wilber ; Garriga, Pere ; Borroto-Escuela, Dasiel O.

Hoboken: Wiley Subscription Services, Inc., A Wiley Company

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