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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.

SUBMITTER: Huang M

PROVIDER: S-EPMC5568435 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Secretory expression and purification of Bacillus licheniformis keratinase in insect cells.

Huang Miaorong M Chen Ruiai R Ren Guangcai G

PloS one 20170823 8

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 ...[more]

PMID: 28832667

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Project description:SUMMARY Research background Haloalkaline proteases are one of the most interesting types of commercial enzymes in various industries due to their high specific activity and stability under extreme conditions. Biochemical characterization of enzymes is an important requirement for determining their potential for application in industrial fields. Most of microbial proteases have been isolated from Bacillus spp. In this study, the purification and characterization of an extracellular haloprotease produced from Bacillus sp. KB111 strain, which was previously isolated from mangrove forest sediments, are investigated for industrial applications. Experimental approach The whole genome of KB111 strain was identified by DNA sequencing. Its produced protease was purified by salting out and anion-exchange chromatography, characterized based on protease activity and stability using a peptide substrate, and identified by LC-MS/MS. Results and conclusions The strain KB111 was identified as Bacillus licheniformis. The molecular mass of its extracellular protease, termed KB-SP, was estimated to be 70 kDa. The optimal pH and temperature for the activity of this protease were 7 and 50 °C, respectively, while the enzyme exhibited maximal activity in the broad salinity range of 2–4 M NaCl. It was fully stable at an alkaline pH range of 7–11 at 50 °C with a half-life of 90 min. Metal ions such as K+, Ca2+ and Mg2+ could enhance the enzyme activity. Therefore, this protease indicates a high potential for the applications in the food and feed industry, as well as the waste management since it can hydrolyse protein at high alkaline pH and salt concentrations. The amino acid profiles of the purified KB-SP determined by LC-MS/MS analysis showed high score matching with the peptidase S8 of B. licheniformis LMG 17339, corresponding to the mature domain of a minor extracellular protease (Vpr). Amino acid sequence alignment and 3D structure modelling of KB-SP showed a conserved catalytic domain, a protease-associated (PA) domain and a C-terminal domain. Novelty and scientific contribution A novel extracellular haloprotease from B. licheniformis was purified, characterized and identified. The purified protease was identified as being a minor extracellular protease (Vpr) and this is the first report on the halotolerance of Vpr. This protease has the ability to work in harsh conditions, with a broad alkaline pH and salinity range. Therefore, it can be useful in various applications in industrial fields.

Dataset Information

Secretory expression and purification of Bacillus licheniformis keratinase in insect cells.

Publications

Secretory expression and purification of Bacillus licheniformis keratinase in insect cells.

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