Secretory expression and purification of Bacillus licheniformis keratinase in insect cells

Abstract

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.

Microbial Proteases Applications

“…The conventional or traditional protein engineering techniques now have thus far employed mutagenesis in the protease domains for modification in the enzymatic properties of proteases. The new approach, termed pro-sequencing protein engineering, is not only an important technique for the study of protein folding mechanisms but also a highly promising technology to create unique proteases that have various beneficial catalytic properties (Hosse et al, 2006; Ruigrok et al, 2011; Mascini et al, 2012; Fang et al, 2016, 2017a; Verma et al, 2016; Huang et al, 2017). The Gly216 is an active site for proteases and is specific to the MA190 mutant from α-lytic proteases.…”

Non-negligible factors in studying the ApoM-S1P axis using EA.hy926 cells

“…C-myc and His tags are widely used in cell experiments (18)(19)(20), such as for the localization of proteins in cellular immunofluorescence assays, and as indicator proteins in Western blots. Exogenous recombinant ApoM with tag proteins could be applied in in vitro studies to ascertain whether ApoM acts as a mediator for the delivery of S1P to S1PRs on the cell membrane, as exogenous ApoM can be directly localized by using antibodies against tag proteins, thereby distinguishing it from endogenous ApoM.…”

Keratinases as Versatile Enzymatic Tools for Sustainable Development

“…Keratinolytic enzymes can be also cloned from newly isolated, often demanding, strains and expressed in a well-known and widely-used heterologous bacterial, e.g., Escherichia coli and B. subtilis , and fungal hosts, such as Pichia pastoris [ 18 , 72 , 130 ]. Heterologous expression of B. lichenifomis PWD-1 keratinase (KerA) have also been reported in insect cells of Spodoptera frugiperda ( Sf9 ) [ 131 ].…”