title: Cloning, Expression, and Functional Characterization of Serine Protease Aprv2 from Virulent Isolate Dichelobacter nodosus of Indian Origin.
abstract: A gene encoding an extracellular protease from Dichelobacter nodosus was characterized and expressed in E. coli rosetta-gami (DE3). The nucleotide sequence analysis revealed an ORF of 1427 bp ecoding 475 amino acids long protein of calculated molecular weight 50.6 kDa and pI value 6.09. The phylogenetic analysis showed relatedness to subtilisin-like serine proteases of peptidase S8 family. The amino acid sequence analysis showed presence of N-terminal pre-peptide (1-23 aa), pro-peptide (24-160 aa), peptidase S8 domain (161-457 aa), and a C-terminal extension (458-475 aa). The gene harboring native signal peptide was expressed in pET-22b(+) for production of AprV2 recombinant protein. SDS-PAGE revealed the highest production of IPTG induced recombinant protein ∼37 kDa at 16 °C after 16 h. The purified protein after Ni-NTA affinity chromatography showed single protein band of ∼37 kDa which was also confirmed by the detection of blue coloured band of same size in Western blotting. The recombinant protein showed activity over broad temperature and pH range with optimum at 35 °C and pH 7.0. Similarly, the enzyme was stable over broad range 15-65 °C and 4-10 pH with maximum stability at 25 °C and pH 6. The activity of purified enzyme was also stimulated in the presence of Ca2+. The purified enzyme showed highest activity towards casein as compared to gelatin and BSA. These findings suggest AprV2 as an important candidate for industrial applications such as pharmaceuticals.
authors.affiliation: Department of Veterinary Microbiology, Dr. G.C. Negi-College of Veterinary and Animal Sciences, CSK-Himachal Pradesh Agricultural University, Palampur, 176062, India.
authors.forename: Aasim Habib
authors.initials: AH
authors.lastname: Wani
authors.affiliation: Department of Veterinary Microbiology, Dr. G.C. Negi-College of Veterinary and Animal Sciences, CSK-Himachal Pradesh Agricultural University, Palampur, 176062, India.
authors.forename: Mandeep
authors.initials: M
authors.lastname: Sharma
authors.affiliation: Department of Veterinary Microbiology, Dr. G.C. Negi-College of Veterinary and Animal Sciences, CSK-Himachal Pradesh Agricultural University, Palampur, 176062, India.
authors.forename: Richa
authors.initials: R
authors.lastname: Salwan
authors.affiliation: Department of Veterinary Physiology and Biochemistry, Dr. G.C. Negi-College of Veterinary and Animal Sciences, CSK-Himachal Pradesh Agricultural University, Palampur, 176062, India.
authors.forename: Geetanjali
authors.initials: G
authors.lastname: Singh
authors.affiliation: Department of Veterinary Microbiology, Dr. G.C. Negi-College of Veterinary and Animal Sciences, CSK-Himachal Pradesh Agricultural University, Palampur, 176062, India.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Chahota
authors.affiliation: Department of Veterinary Microbiology, Dr. G.C. Negi-College of Veterinary and Animal Sciences, CSK-Himachal Pradesh Agricultural University, Palampur, 176062, India. sverma8@gmail.com.
authors.forename: Subhash
authors.initials: S
authors.lastname: Verma
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D020376:Dichelobacter nodosus / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity*; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007194:India; D008958:Models, Molecular; D010802:Phylogeny; D011994:Recombinant Proteins / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D057057:Serine Proteases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D057057:Serine Proteases
keywords: Characterization; Dichelobacter nodosus; Expression; Protease; Virulence