Anti-FLAG tag antibody [HRP]

Recombinant proteins have increased our knowledge regarding the physiological role of proteins; however, affinity purification tags are often not cleaved prior to analysis, and their effects on protein structure, stability and assembly are often overlooked. In this study, the stabilizing effects of an N-terminus dual-FLAG (FT2) tag fusion to transthyretin (TTR), a construct used in previous studies, are investigated using native ion mobility-mass spectrometry (IM-MS). A combination of collision-induced unfolding and variable-temperature electrospray ionization is used to compare gas- and solution-phase stabilities of FT2-TTR to wild-type and C-terminal tagged TTR Despite an increased stability of both gas- and solution-phase FT2-TTR, thermal degradation of FT2-TTR was observed at elevated temperatures, viz., backbone cleavage occurring between Lys9 and Cys10. This cleavage reaction is consistent with previously reported metalloprotease activity of TTR [Liz et al. 2009] and is suppressed by either metal chelation or excess zinc. This study brings to the fore the effect of affinity tag stabilization of TTR and emphasizes unprecedented detail afforded by native IM-MS to assess structural discrepancies of recombinant proteins from their wild-type counterparts.

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.