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™-1 donor vector by BamHI/HindIII restriction sites. The constructed vector was transformed to E. coli DH10Bac™ 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. Ta... More
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™-1 donor vector by BamHI/HindIII restriction sites. The constructed vector was transformed to E. coli DH10Bac™ 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 (SDS-PAGE) 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°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.
