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Cloning, Expression, and Functional Characterization of Serine Protease Aprv2 from Virulent Isolate Dichelobacter nodosus of Indian Origin

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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.

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References

  1. Bennett, G. N., & Hickford, J. G. H. (2011). Ovine footrot: New approaches to an old disease. Veterinary Microbiology, 148, 1–7.

    Article  CAS  Google Scholar 

  2. Kennan, R. M., Gilhuus, M., Frosth, S., Seemann, T., Dhungyel, O. P., Whittington, R. J., Boyce, J. D., Powell, D. R., Aspan, A., Jorgensen, H. J., Bulach, D. M., & Rooda, J. I. (2014). Genomic evidence for a globally distributed. Bimodal Population in the Ovine Footrot Pathogen Dichelobacter nodosus. mBio, 5(5), e01821–14.

    CAS  Google Scholar 

  3. Egerton, J. R., Roberts, D. S., & Parsonson, I. M. (1969). The aetiology and pathogenesis of ovine footrotA histological study of the bacterial invasion. Journal of Comparative Pathology, 79, 209–216.

    Article  Google Scholar 

  4. Stewart, D. J., Clark, B. L., & Jarret, R. G. (1984). Difference between strains of Bacteroides nodosus in their effects on the severity of footrot, bodyweight and wool growth on Merino sheep. Australian Veterinary Journal, 61, 349–352.

    Article  Google Scholar 

  5. Ghosh, A., Chakrabarti, K., & Chattopadhyay, D. (2009). Cloning of feather-degrading minor extracellular protease from Bacillus cereus DCUW: dissection of the structural domains. Microbiology, 155, 2049–57.

    Article  CAS  Google Scholar 

  6. Arnorsdottir, J., Smaradottir, R. B., Magnusson, O. T., Thorbjarnardottir, S. H., Eggertsson, G., & Kristjansson, M. (2002). Characterization of a cloned subtilisin-like serine proteinase from a psychrotrophic Vibrio species. European Journal of Biochemistry, 269, 5536–5546.

    Article  CAS  Google Scholar 

  7. Kwon, K., Hasseman, J., Latham, S., Grose, C., Do, Y., Fleischmann, R. D., Pieper, R., & Petersen, S. N. (2011). Recombinant expression and functional analysis of proteases from Streptococcus pneumoniae, Bacillus anthracis and Yersinia pestis. BMC Biochemistry, 12, 17.

    Article  CAS  Google Scholar 

  8. Larsen, A. N., Moe, E., Helland, R., Gjellesvik, D. R., & Willassen, N. P. (2006). Characterization of a recombinantly expressed proteinase K-like enzyme from a psychrotrophic Serratia sp. FEBS Journal, 273, 47–60.

    Article  CAS  Google Scholar 

  9. Maurer, K. H. (2004). Detergent proteases. Current Opinion in Biotechnology, 15, 330–334.

    Article  CAS  Google Scholar 

  10. Craik, C. S., Page, M. J., & Madison, E. L. (2011). Proteases as therapeutics. Biochemical Journal, 435, 1–16.

    Article  CAS  Google Scholar 

  11. Chanalia, P., Gandhi, D., Jodha, D., & Singh, J. (2011). Applications of microbial proteases in pharmaceutical industry: an overview. Reviews in Medical Microbiology, 22, 96–101.

    Article  Google Scholar 

  12. Shen, H. B., & Chou, K. C. (2009). Identification of proteases and their types. Analytical Biochemistry, 385, 153–160.

    Article  CAS  Google Scholar 

  13. Skerman, T. M. (1975). Determination of some in vitro growth requirements of Bacteroides nodosus. Journal of General Microbiology, 87, 107–119.

    Article  CAS  Google Scholar 

  14. La Fontaine, S., Egerton, J. R., & Rood, J. I. (1993). Detection of Dichelobacter nodosus using species specific oligonucleotides as PCR primers. Veterinary Microbiology, 35, 101–117.

    Article  Google Scholar 

  15. Dhungyel, O. P., Whittington, R. J., & Egerton, J. R. (2002). Serogroup specific single and multiplex PCR with pre-enrichment culture and immuno-magnetic bead capture for identifying strains of D. nodosus in sheep with footrot prior to vaccination. Molecular and Cellular Probes, 16, 285–296.

    Article  CAS  Google Scholar 

  16. Cheetham, B. F., Tanjung, L. R., Sutherland, M., Druitt, J., Green, G., McFarlane, J., Bailey, G. D., Seaman, J. T., & Katz, M. E. (2006). Improved diagnosis of virulent footrot using intA gene. Veterinary Microbiology, 116, 166–174.

    Article  CAS  Google Scholar 

  17. Palmer, M. A. (1993). A gelatin test to detect activity and stability of proteases produced by Dichelobacter (Bacteroides) nodosus. Veterinary Microbiology, 36, 113–122.

    Article  CAS  Google Scholar 

  18. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403–410.

    Article  CAS  Google Scholar 

  19. Letunic, C. R. R., Pils, B., Pinkert, S., Schultz, J., & Bork, P. (2006). SMART 7: recent updates to the protein domain annotation resource. Nucleic Acids Research, 34, D257–260.

    Article  CAS  Google Scholar 

  20. Marchler-Bauer, A., Anderson, J. B., Cherukuri, P. F., DeWeese-Scott, C., Geer, L. Y., He, M. G. S., Hurwitz, D. I., Jackson, J. D., Ke, Z., Lanczycki, C. J., Liebert, C. A., Liu, C., Lu, F., Marchler, G. H., Mullokandov, M., Shoemaker, B. A., Simonyan, V., Song, J. S., Thiessen, P. A., Yamashita, R. A., Yin, J. J., Zhang, D., & Bryant, S. H. (2005). CDD: a Conserved Domain Database for protein classification. Nucleic Acids Research, 33, 192–196.

    Article  Google Scholar 

  21. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance and maximum parsimony methods. Molecular Biology and Evolution, 28, 2731–2739.

    Article  CAS  Google Scholar 

  22. Schwede, T., Kopp, J., Guex, N., & Peitsch, M. C. (2003). SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Research, 31, 3381–3385.

    Article  CAS  Google Scholar 

  23. Benkert, P., Tosatto, S. C. E., & Schomburg, D. (2008). QMEAN: A comprehensive scoring function for model quality assessment. Proteins: Structure, Function, and Bioinformatics, 71(1), 261–277.

    Article  CAS  Google Scholar 

  24. Folin, O., & Ciocalteu, V. (1927). On tyrosine and tryptophane determinations in proteins. Journal of Biological Chemistry, 73, 627–650.

    CAS  Google Scholar 

  25. Wang, Q., Miao, J. L., Hou, Y. H., Ding, Y., Wang, G. D., & Li, G. Y. (2005). Purification and characterization of an extracellular cold–active serine protease from the psychrophilic bacterium Colwellia sp. NJ341. Biotechnology Letters, 27, 1195–1198.

    Article  CAS  Google Scholar 

  26. Rao, C. S., Sathish, T., Brahamaiah, P., Kumar, T. P., & Prakasham, R. S. (2009). Development of a mathematical model for Bacillus circulans growth and alkaline protease production kinetics. Journal of Chemical Technology and Biotechnology, 84, 302–307.

    Article  CAS  Google Scholar 

  27. Kennan, R. M., Wong, W., Dhungyel, O. P., Han, X., Wong, D., Parker, D., Rosado, C. J., Law, R. H., McGowan, S., Reeve, S. B., Levina, V., Powers, G. A., Pike, R. N., Bottomley, S. P., Smith, A. I., Marsh, I., Whittington, R. J., Whisstock, J. C., Porter, C. J., & Rood, J. I. (2010). The Subtilisin-like protease AprV2 is required for virulence and uses a novel disulphide-tethered exosite to bind substrates. PLoS Pathogens, 6(11), E1001210.

    Article  Google Scholar 

  28. Myers, G. S., Parker, D., Al-Hasani, K., Kennan, R. M., Seemann, T., Ren, Q., Badger, J. H., Selengut, J. D., Deboy, R. T., Tettelin, H., Boyce, J. D., McCarl, V. P., Han, X., Nelson, W. C., Madupu, R., Mohamoud, Y., Holley, T., Fedorova, N., Khouri, H., Bottomley, S. P., Whittington, R. J., Adler, B., Songer, J. G., Rood, J. I., & Paulsen, I. T. (2007). Genome sequence and identification of candidate vaccine antigens from the animal pathogen Dichelobacter nodosus. Nature Biotechnology, 25(5), 569–575.

    Article  CAS  Google Scholar 

  29. Riffkin, M. C., Wang, L. F., Kortt, A. A., & Stewart, D. J. (1995). A single amino-acid change between the antigenically different extracellular serine proteases V2 and B2 from Dichelobacter. Gene, 167(1–2), 279–283.

    Article  CAS  Google Scholar 

  30. Deng, A., Wu, J., Zhang, G., & Wen, T. (2011). Molecular and structural characterization of a surfactant-stable high-alkaline protease AprB with a novel structural feature unique to subtilisin family. Biochimie, 93, 783–791.

    Article  CAS  Google Scholar 

  31. VanderLaan, J. M., Teplyakov, A. V., Kelders, H., Kalk, K. H., Misset, O., Mulleners, L. J., & Dijkstra, B. W. (1992). Crystal structure of the high-alkaline serine protease PB92 from Bacillus alcalophilus. Protein Engineering, 5(5), 405–411.

    Article  CAS  Google Scholar 

  32. Wandersman, C. (1989). Secretion, processing and activation of bacterial extracellular proteases. Molecular Microbiology, 3(12), 1825–1831.

    Article  CAS  Google Scholar 

  33. Wong, W., Kennan, R. M., Rosado, C. J., Rood, J. I., Whisstocka, J. C., & Portera, C. J. (2010). Crystallization of the virulent and benign subtilisin-like proteases from the ovine footrot pathogen Dichelobacter nodosus. Acta Crystallographica Section, F66, 289–293.

    Google Scholar 

  34. Rawlings, N. D., & Barett, A. J. (1994). Families of serine peptidases. Methods in Enzymology, 224, 19–61.

    Article  Google Scholar 

  35. Ather, A. (2009). Identification, cloning and expressions of proteases from a cold adapted organism Aliivibrio salmonicida KJE-3900. University of Tromso.

  36. Vaughan, P. R., Wang, L. F., Stewart, D. J., Lilley, G. G., & Kortt, A. A. (1994). Expression in Escherichia coli of the extracellular basic protease frorn Dichelobacter nodosus. Microbiology, 140, 2093–2100.

    Article  CAS  Google Scholar 

  37. Sarvas, M., Harwood, C. R., Bron, S., & Dijl, J. M. (2004). Post-translocational folding of secretory proteins in Gram-positive bacteria. Biochimica et Biophysica Acta, 1694, 311–327.

    CAS  Google Scholar 

  38. Godde, C., Sahm, K., Brouns, S. J. J., Kluskens, L. D., Oost, J. V., deVos, W. M., & Antranikian, G. (2005). Cloning and expression of islandisin, a new thermostable subtilisin from Fervidobacterium islandicum, in Escherichia coli. Applied and Environmental Microbiology, 71(7), 3951–3958.

    Article  Google Scholar 

  39. Chen, X. L., Xie, B. B., Lu, J. T., He, H. L., & Zhang, Y. Z. (2007). A novel type of subtilase from the psychrotolerant bacterium Pseudoalteromonas sp. SM9913: catalytic and structural properties of deseasin MCP-01. Microbiology, 153, 2116–2125.

    Article  CAS  Google Scholar 

  40. Huston, A. L., Methe, B., & Deming, J. W. (2004). Purification, characterization, sequencing of an extracellular cold-active aminopeptidase produced by marine psychrophile Colwellia psychrerythraea strain 34H. Applied and Environmental Microbiology, 70, 2321–2328.

    Article  Google Scholar 

  41. Kortt, A. A., Caldwell, J. B., Lilley, G. G., Edwards, R., Vaughan, J., & Stewart, D. J. (1994). Characterization of a basic serine protease (pI ∼ 9.5) secreted by virulent strains of Dichelobacter nodosus and identification of a distinct, but closely related, proteinase secreted by benign strains. Journal of Biochemistry, 299, 521–525.

    Article  CAS  Google Scholar 

  42. Setyorini, E., Kim, Y. J., Takenaka, S., Murakami, S., & Aoki, K. (2006). Purification and characterization of a halotolerant intracellular protease from Bacillus subtilis strain FP-133. Journal of Basic Microbiology, 46(4), 284–304.

    Article  Google Scholar 

  43. Yan, B. Q., Chen, X. L., Hou, X. Y., He, H., Zhou, B. C., & Zhang, Y. Z. (2009). Molecular analysis of the gene encoding a cold-adapted halophilic subtilase from deep-sea psychrotolerant bacterium Pseudoalteromonas sp. SM9913: cloning, expression, characterization and function analysis of the C-terminal PPC domains. Extremophiles, 13, 725–733.

    Article  CAS  Google Scholar 

  44. Pushpam, P. L., Rajesh, T., & Gunasekaran, P. (2011). Identification and characterization of alkaline serine protease from goat skin surface metagenome. AMB Express, 1, 3.

    Article  Google Scholar 

  45. Zhang, Y., Zhao, J., & Zeng, R. (2011). Expression and characterization of a novel mesophilic protease from metagenomic library derived from Antarctic coastal sediment. Extremophiles, 15, 23–29.

    Article  Google Scholar 

  46. Doddapaneni, K. K., Tatineni, R., Potumarthi, R., & Mangamoori, L. N. (2007). Optimization of media constituents through response surface methodology for improved production of alkaline proteases by Serratia rubidaea. Journal of Chemical Technology and Biotechnology, 82, 721–729.

    Article  CAS  Google Scholar 

  47. Morita, Y., Hasan, Q., Sakaguchi, T., Murakami, Y., Yokoyama, K., & Tamiya, E. (1998). Properties of a cold-active protease from psychrotrophic Flavobacterium balustinum P104. Applied Microbiology and Biotechnology, 50(6), 669–675.

    Article  CAS  Google Scholar 

  48. Secades, P., Alvarez, B., & Guijarro, J. A. (2001). Purification and characterization of a psychrophilic calcium induced, growth-phase dependent metalloprotease from the fish pathogen Flavobacterium psychrophilum. Applied and Environmental Microbiology, 67(6), 2436–2444.

    Article  CAS  Google Scholar 

  49. Tariq, A. L., Reyaz, A. L., & Prabakaran, J. J. (2011). Purification and Characterization of 56 KDa cold active protease from Serratia marcescens. African Journal of Microbiology Research, 5(32), 5841–5847.

    Article  CAS  Google Scholar 

  50. Shankar, S., Rao, M., & Laxman, R. S. (2011). Purification and characterization of an alkaline protease by a new strain of Beauveria sp. Process Biochemistry, 46, 579–585.

    Article  CAS  Google Scholar 

  51. Srilakshmi, J., Madhavi, J., Lavanya, S., & Ammani, K. (2015). Commercial potential of fungal protease: past, present and future prospects. Journal of Pharmaceutical, Chemical and Biological Sciences, 2(4), 218–234.

    CAS  Google Scholar 

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Acknowledgments

The authors acknowledge the financial support received from the ICAR under NAIP project on footrot. The authors also acknowledge CSK-Himachal Pradesh Agricultural University, Palampur, for logistic support.

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  1. Department of Veterinary Microbiology, Dr. G.C. Negi-College of Veterinary and Animal Sciences, CSK-Himachal Pradesh Agricultural University, Palampur, 176062, India

    Aasim Habib Wani, Mandeep Sharma, Richa Salwan, Rajesh Chahota & Subhash Verma

  2. Department of Veterinary Physiology and Biochemistry, Dr. G.C. Negi-College of Veterinary and Animal Sciences, CSK-Himachal Pradesh Agricultural University, Palampur, 176062, India

    Geetanjali Singh

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  1. Aasim Habib Wani
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Wani, A.H., Sharma, M., Salwan, R. et al. Cloning, Expression, and Functional Characterization of Serine Protease Aprv2 from Virulent Isolate Dichelobacter nodosus of Indian Origin. Appl Biochem Biotechnol 180, 576–587 (2016). https://doi.org/10.1007/s12010-016-2117-5

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