Acknowledgements We thank Moshe Mevarech for the plasmid pWL-CBD and Valery Tarasov for the plasmid pVT. We thank Stefan Streif for critical reading of the manuscript and helpful comments, and Friedhelm Pfeiffer for help with implementing the database infrastructure into HaloLex. This work was supported by European Union FP6 INTERACTION PROTEOME (Grant No. LSHG-CT-2003-505520). Electronic supplementary material Additional file 1: Expression of the CBD-tagged bait protein and the untagged control. A, B Schematic representation of the bait-CBD learn more expression
vector pMS4 and the corresponding bait-control pMS6. Both plasmids contain a pUC origin (not indicated) and an ampicillin resistance (AmpR) for amplification in E. coli. The novobiocin resistance (NovR) and β-galactosidase (bgaH) are for selection of transformants in Hbt. salinarum. Bait genes are cloned between the attR1 and attR2 sites via Gateway recombination (Invitrogen). Between the bait protein and the CBDs (pMS4) or the
His-Tags (pMS6) is a short linker sequence (IGAVEER, the linker of the two β-sheets in Hbt. salinarum dodecin). Downstream of the fusion protein is a transcriptional terminator from the Hbt. salinarum bop gene (not shown). C, D The plasmids do not contain a haloarchaeal origin of replication. After transformation into Hbt. salinarum, they are integrated into the genome at the site of the bait protein by homologous recombination. C Integration of Mdivi1 pMS4 constructs (red) into the genome (blue) leads to the expression of the bait C-terminally fused to CBD under control of the bait’s endogenous promoter and the expression of an N-terminal bait-CBD fusion under control of the promoter PrR16 (a highly active, modified ferredoxin promoter [118, 119]). D Integration of pMS6 Protein kinase N1 constructs results in similar promoter-bait constructs without CBD. (PDF 43 KB) Additional file 2: Details
on result evaluation of the bait fishing experiments. (PDF 87 KB) Additional file 3: Protein identifications in bait fishing experiments. (XLS 1 MB) Additional file 4: Identification of the core signaling proteins in all bait fishing experiments. The numbers show the sequence coverage of the protein identification. Numbers in bold type indicate that this protein was identified as an interaction partner by the SILAC ratio. Numbers in italics indicate that this prey was identified with relatively high sequence coverage in a one-step bait fishing experiment but the SILAC ratio was close to one and that this prey was identified as an interaction partner in two-step bait fishing. Together, this indicates a dynamic interaction between bait and prey. (PDF 41 KB) Additional file 5: Bait fishing experiments for the Che interaction network. The upper part of the table shows the initial experiments with the 10 Hbt. salinarum Che proteins known before the start of this study. The lower part lists experiments with baits which were identified as interaction selleck compound partners in the initial experiments.