IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Screening of yeast and bacterial expression of SLC35A3 transporters based on GFP technology
Autor/es:
FAVAROLO B, SALDAÑA C, PEPPE S, ZARYCKI M, FUENTEALBA J, ESERSKY I, BREDESTON L
Lugar:
Villa Carlos Paz, Cordoba
Reunión:
Congreso; XLII Reunión Anual de la Sociedad Argentina de Biofísica; 2013
Institución organizadora:
Sociedad Argentina de Biofisica (SAB)
Resumen:
Nucleotide-sugar transporters (NST, SLC35 family) control the flux of activated sugar to the lumen of Golgi apparatus, where glyconjugate biosynthesis take place. To these transport process an antiport mechanism, nucleotide-sugar/nucleoside monophosphate, have been proposed. The main limitation to structure-function studies of NST is the production of high levels of protein. Recently was described a methodology, based on GFP fusion to membrane proteins, for a rapid screening of expression and purification conditions. In this work we studied the expression of UDP-GlcNac-SLC35A3 transporters from diverse species tagged to GFP in three different systems, P. pastoris, S. cerevisiae and E. coli. In S. cerevisiae, C03H5.2 the C. elegans A3-homolog, showed the higher expression levels (in compassion to mouse-A3 and Xenopus-A3) measuring by fluorescence in whole cells and quantified by a purified yEGFP standard (2-3 mg C03H5.2-GFPhis8/L of culture). Preliminary attempts to purified the protein showed a yield of 1.1 mg/L culture. Expression of mouse SLC35A3-GFP in Pichia pastoris showed similar levels to S. cerevisiae in an initial screening. The advantage of this system is the obtention of stable transformants and multiple insertion events (that occurs spontaneously at about 1-10% of single insertion). Now we are screening a large number of clones looking a small number of highly expressing clones. Finally expression of mouse SLC35A3-GFP in E. coli showed slight expression than yeast under initial conditions. Optimization of IPTG concentration, temperature and time could be important to improve the final yields.