The cloned product was used to transform a S cerevisiae strain Y

The cloned product was used to transform a S. cerevisiae strain Y187 (ΔTRP1). A cDNA library was constructed with RNA from P. brasiliensis yeast cells and cloned in the expression vector pGADT7-Rec by using the Matchmaker™Library Construction

& Screening (Clontech Laboratories, Inc) [36]. The pGADT7-Rec vector contains LEU2 gene, allowing the selection in minimal medium without leucine and a GAL4 DNA-activation domain. The cloned products were transformed in S. cerevisiae strain AH109 (Δ LEU2). The Y187 strain containing pGBK-T7-PbSP was used to screen the pGADT7-Rec library transformed in AH109 strain by yeast mating. The positive this website interactions activate the transcription of ADE2, HIS3 and MEL1 genes,

which allows the selection in minimal medium without tryptophan, leucine, adenine and histidine. Minimal medium without these amino acids and containing X-alpha-GAL also confirms the activation of the transcription of the MEL1 gene. The PbSP baited clones were amplified by using AD-LD 5′ (5′-CTATTCGATGATGAAGATACCCCACCAAACCC-3′) and AD-LD 3′ (5′-GTGAACTTGCGGGGTTTTTCAGTATCTACGATT-3′) oligonucleotides for pGADT7-Rec and sequenced as described above. The positive interactions were confirmed by using the in vitro translation system TNT® T7 Coupled Reticulocyte Lysate Systems (Promega Corporation) with S35 methionine and Wnt inhibitor coimmunoprecipitation of the translated proteins (Matchmaker™ Co-IP Kit, Clontech Laboratories, Inc). Briefly, the translated serine protease fused to c-myc epitope (c-myc-SP) and the translated proteins fused to hemaglutinin SPTLC1 epitope (HA-Prey) were mixed at 25°C for 1 h. The mixture was incubated with protein A Agarose beads and with the monoclonal c-myc antibody in PBS at 25°C for 1 h. After washing, the beads containing proteins were resuspended in SDS-loading buffer [50 mM Tris-HCl, pH 6.8; 100 mM dithiothreitol,

2% (w/v) SDS; 0.1% (w/v) bromophenol blue; 10% (v/v) glycerol], followed by boiling at 80°C for 5 min. The proteins were separated on a SDS-PAGE 4-12% linear gradient. The gel was fixed with 20% (v/v) ethanol and 10% (v/v) acetic acid for 30 min, and incubated in 20 mL of fluorographic reagent NAMP 100 (Amplify Fluorographic Reagent – GE Healthcare®). The gels were dried at 80°C for 90 min under vacuum and autoradiography was obtained. Controls were performed. Each assay was repeated three times with a different batch of in vitro translated product to confirm the results. Acknowledgements This research was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq grants 472947/2007-9 and 558405/2008-8), Coordenação de Aperfeiçoamento de Ensino Superior (CAPES), Financiadora de Estudos e Projetos (FINEP, grants 0106121200 and 010477500), Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG) and Secretaria de Estado de Ciência e Tecnologia de Goiás (SECTEC-GO).

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