Anti-FLAG tag antibody, clone O4 [SureLight® P3]

The keratinase (kerA) gene from Bacillus licheniformis PWD-1 was expressed and purified in insect cells. First, the sequence encoding Ker-His-Flag was designed based on the amino acid sequence of the protein and peptide and codon optimization in order to ensure the high expression in insect cells. In the next step, the synthetic DNA was inserted into the pUC57 vector and then sub-cloned in the pFastBac (TM)-1 donor vector by BamHI/ HindIII restriction sites. The constructed vector was transformed to E. coli wDH10Bac (TM) cell to generate recombinant bacmid carrying Ker-His-Flag. Recombinant viruses were produced by infecting insect Spodoptera frugiperda (Sf9) cells with bacmid DNA and used for proteins production. Target proteins were purified from the cell supernatants by Ni2+-NTA affinity chromatography and evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and western blot. The purified product contained two peptides with molecular weights of 38 kDa and 30 kDa and had an optimal pH and temperature at 8.0 and 45 degrees C for keratinolytic activity, respectively. The final product had a specific activity of about 635 U/mg. In summary, we have demonstrated that the open reading frame containing recombinant Ker-His-Flag was expressed and secreted by leader peptide of mellittin from Apis mellitera in insect cells and affinity purification through 8His-Flag tag. It presents an alternative technology for producing keratinases. To our knowledge, it was the first report on the expression of functional keratinase from Bacillus licheniformis in insect cells system.

RNA binding proteins, important players in post-transcriptional gene regulation, usually exist in ribonuclear complexes. However, even in model systems like Arabidopsis characterisation of RBP associated proteins is limited. In this study, we investigated the interacting proteins of the Arabidopsis AtRBP45b, which is involved in stress signalling. In vivo localisation of AtRBP45b was conducted using 35S-GFP. FLAG-tagged AtRBP45b under control of the 35S promoter in the Atrbp45b-1 mutant background was used to pull down AtRBP45b interacting proteins. Yeast two-hybrid analysis, fluorescence energy resonance transfer assays were used to confirm the veracity of the AtRBP45b interacting proteins. In planta GFP-tagging indicated AtRBP45b is localised to the nucleus and the cytosol.AtRBP45b protein has a N-terminal proline-rich region and a C-terminal glutamine-rich domain that are usually involved in protein-protein interactions. Co-immunoprecipitation followed by mass spectrometry-based protein sequencing led to identification of 30 proteins that interacted with AtRBP45b. Using information from interactome databases (BIOGRID, INTACT and STRING), pull-down assays and localisation data, 12 putative interacting proteins were selected for yeast two-hybrid analysis. Cap-binding protein (CBP20, At5g44200) and polyA-binding protein (PAB8, At1g49760) were shown to interact with AtRBP45b. Based on its interacting partners we speculate that AtRBP45b may play an important role in RNA metabolism, especially in aspects related to mRNA stability and translation initiation during stress conditions in plants.