Synthetic Gene-Based Heterologous Expression, Proteolytic, and Structural Characterization of Caseinolytic Protease of Lactobacillus plantarum IIA-1A5
Abstract
Genome sequence of Indonesian probiotic of Lactobacillus plantarum II1A5 contains a gene encoding a proteolytic subunit of caseinolytic protease, designated as ClpP_LP. This study aims to express the Clp gene heterological and apply its proteolytic activity to some livestock products. To address this, the gene encoding ClpP_LP was optimized in silico by improving its Codon Adaptation Index and GC content to 0.94 and 53.62%, respectively. The optimized gene was then inserted into pET28a, transformed into Escherichia coli BL21(DE3), and over-expressed by induction of 1 mM Isopropyl β-D-1-thiogalactopyranoside at 37°C. The result showed that ClpP_LP was successfully over-expressed in a fully soluble form with the specific activity towards milk casein was 7739.89 AU mg-1. This activity was significantly greater than that of chymotrypsin. Further, the three-dimensional model of ClpP_LP was built using SWISS MODEL, which showed that this protein formed a homo-tetradecameric (14-mer) structure with each monomer consisting of 7 α-helix and 10 β-sheets. The identification of the active side showed that the active side of ClpP_LP is Ser-97, His-122, Asp-171, and forms a substrate-binding cavity with a size of about 29.5 Ǻ. Overall, our approach can serve as an appropriate platform for the production of ClpP_LP in a large-scale production for various applications in dairy products and derivatives.
References
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Arief, I. I., B. S. L. Jenie, M. Astawan, K. Fujiyama, & A. B. Witarto. 2015. Identification and probiotic characteristics of lactic acid bacteria isolate from Indonesian local beef. Asian J. Anim. Sci. 9:25-36. https://doi.org/10.3923/ajas.2015.25.36
Arief, I. I., Jakaria, T. Suryati, Z. Wulandari, & E. Andreas. 2013. Isolation and characterization of plantaricin produced by Lactobacillus plantarum Strain (IIA-1A5, IIA-1B1, IIA-2B2). Med. Pet. 36:91-100. https://doi.org/10.5398/medpet.2013.36.2.91
Aruna, K., J. Shah, & R. Birmole. 2014. Production and partial char-acterization of alkaline protease from Bacillus tequilensis strains CSGAB 0139 isolated from spoilt cottage cheese. Int. J. Appl. Biol. Pharm. 5:201-21.
Ban, E. & C. R. Picu. 2013. Strength of DNA sticky end links. Biomacromolecules 15:143−149. https://doi.org/10.1021/bm401425k
Biasini, M., S. Bienert, A. Waterhouse, K. Arnold, G. Studer, T. Schmidt, F. Kiefer, T. Gallo Cassarino, M. Bertoni, & L. Bordoli. 2014. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42: W252-W258. https://doi.org/10.1093/nar/gku340
Bienert, S., A. Waterhouse, T. A. P. de Beer, G. Tauriello, G. Studer, L. Bordoli, & T. Schwede. 2017. The SWISS-MODEL repository--new features and functionality. Nucleic Acids Res. 45:D313-D319. https://doi.org/10.1093/nar/gkw1132
Bordoli, L., F. Kiefer, K. Arnold, P. Benkert, J. Battey, & T. Schwede. 2009. Protein structure homology modeling using SWISS-MODEL workspace. Nat. Protoc. 4:1-13. https://doi.org/10.1038/nprot.2008.197
Browning, D. F., R. E. Godfrey, K. L. Richards, C. Robinson, & S. J. W. Busby. 2019. Exploitation of the Escherichia coli lac operon promoter for controlled recombinant protein production. Biochem. Soc. Trans. 47:755–763. https://doi.org/10.1042/BST20190059
Budiman, C., I. I. Arief, F. Opook, & M. Yusuf. 2021. A meat-derived lactic acid bacteria, Lactobacillus plantarum IIA, expresses a functional parvulin-like protein with unique structural property. Online J. Biol. Sci. 21:120-135. https://doi.org/10.3844/ojbsci.2021.120.135
Camacho, C., G. Coulouris, V. Avagyan, N. Ma, J. Papadopoulos, K. Bealer, & T. L. Madden. 2009. BLAST+: architecture and applications. BMC Bioinformatics. 10, 421. https://doi.org/10.1186/1471-2105-10-421
Cardoso, V. M., G. Campani, M. P. Santos, G. G. Silva, M. C. Pires, V. M. Gonçalves, R. de C. Giordano, C. R. Sargo, A. C. L. Horta, & T. C. Zangirolami. 2020. Cost analysis based on bioreactor cultivation conditions: Production of a soluble recombinant protein using Escherichia coli BL21(DE3). Biotechnol. Rep. 26:1-13. https://doi.org/10.1016/j.btre.2020.e00441
Fatmarani, R., I. I. Arief, & C. Budiman. 2018. Purification of bacteriocin from Lactobacillus plantarum IIA-1A5 grown in various whey cheese media under freeze dried condition. Trop. Anim. Sci. J. 41:191-199. https://doi.org/10.5398/tasj.2018.41.1.53
Fiege, K., & N. F. Dinkel. 2020. Construction of a new T7 promoter compatible Escherichia coli Nissle 1917 strain for recombinant production of heme dependent proteins. Microb. Cell Fact. 19:190. https://doi.org/10.1186/s12934-020-01447-5
Florentin, A., D. R. Stephens, C. F. Brooks, R. P. Baptista, & V. Muralidharan. 2020. Plastid biogenesis in malaria parasites requires the interactions and catalytic activity of the Clp proteolytic system. PNAS. https://doi.org/10.1073/pnas.1919501117
Frees, D., U. Gerth, & H. Ingmer. 2014. Clp chaperones and proteases are central in stress survival, virulence and antibiotic resistance of Staphylococcus aureus. Int. J. Med. Microbiol. 304:142-149. https://doi.org/10.1016/j.ijmm.2013.11.009
Fu, H., Y. Liang, X. Zhong, Z. Pan, L. Huang, H. Zhang, Y. Xu, W. Zhou, & Z. Liu. 2020. Codon optimization with deep learning to enhance protein. Sci. Rep. 10: Article number 17617. https://doi.org/10.1038/s41598-020-74091-z
Gaspar, P., J. L. Oliveira, J. Frommlet, M. A. Santos, & G. Moura. 2012. EuGene: maximizing synthetic gene design for heterologous expression. Bioinformatics 28:683-2684. https://doi.org/10.1093/bioinformatics/bts465
Gurung, N., S. Ray, S. Bose, & V. Rai. 2013. A broader view: Microbial enzymes and their relevance in industries, medicine, and beyond. Biomed Res. Int. 2013:1-18. https://doi.org/10.1155/2013/329121
Haddad, Y., V. Adam, & Z. Heger. 2020. Ten quick tips for homology modeling of highresolution protein 3D structures. Plos Comput. Biol. 16:e1007449. https://doi.org/10.1371/journal.pcbi.1007449
Hughes, R. A., A. E. Miklos, & A. D. Ellington. 2011. Gene synthesis: methods and applications. Methods Enzymol. 498:277-309. https://doi.org/10.1016/B978-0-12-385120-8.00012-7
Kang, Y. S., J. A. Song, K. Y. Han, & J. Lee. 2015. Escherichia coli EDA is a novel fusion expression partner to improve solubility of aggregation-prone heterologous proteins. J. Biotechnol. 194:39-47. https://doi.org/10.1016/j.jbiotec.2014.11.025
Kuhlman, B. & P. Bradley. 2019. Advances in protein structure prediction and design. Nat. Rev. Mol. Cell Biol. 20:681-697. https://doi.org/10.1038/s41580-019-0163-x
Lee, B. G., M. K. Kim, & H. K. Song. 2011. Structural insights into the conformational diversity of ClpP from Bacillus subtilis. Mol. Cells. 32:589-595. https://doi.org/10.1007/s10059-011-0197-1
Li, Q., L, Yi, P. Marek, & B. L. Iverson. 2013. Commercial proteases: Present and future. FEBS Lett. 587:1155-1163. https://doi.org/10.1016/j.febslet.2012.12.019
Liu, B., Q. Kong, D. Zhang, & L. Yan. 2018. Codon optimization significantly enhanced the expression of human 37-kDa iLRP in Escherichia coli. 3 Biotech 8:Article number 210. https://doi.org/10.1007/s13205-018-1234-y
Mauro, V. P. 2018. Codon optimization in the production of recombinant biotherapeutics: Potential risks and considerations. BioDrugs 32:69-81. https://doi.org/10.1007/s40259-018-0261-x
Miguel, Â. S. M., T. S. Martins-Meyer, E. Veríssimo da Costa Figue-iredo, B. W. P. Lobo, & G. M. Dellamora-Ortiz. 2013. Enzymes in bakery: Current and future trends. In: Muzzalupo I (Eds). Food Industry. InTech, Rijeka, Croatia.
Murwantoko., C. K. Fusianto, & Triyanto. 2016. Gene cloning and protein expression of koi herpesvirus ORF25. Hayati 23:143-149. https://doi.org/10.1016/j.hjb.2016.10.001
Newman, M., T. Strzelecka, L. F. Dorner, Schildkraut, & A. K. Aggarwal. 1994. Structure of endonuclease BamH1 and its relationship to EcoR1. Nature 368:660-664. https://doi.org/10.1038/368660a0
Nigam, P. S. 2013. Microbial enzymes with special characteristics for biotechnological applications. Biomolecules 3:597-611. https://doi.org/10.3390/biom3030597
Rahimzadeh, M., M. Sadeghizadeh, F. Najafi, S. S. Arab, & H. Mobasheri. 2016. Impact of heat shock step on bacterial transformation efficiency. Mol. Biol. Res. Commun. 5:257-261.
Rahmen, N., C. D. Schlupp, H. Mitsunaga, A. Fulton, T. Aryani, L. Esch, U. Schaffrath, E. Fukuzaki, K. E. Jaeger, & J. Büchs. 2015. A particular silent codon exchange in a recombinant gene greatly influences host cell metabolic activity. Microb. Cell. Fact. 14:156. https://doi.org/10.1186/s12934-015-0348-8
Ramirez, O., R. Zamora, G. Espinosa, E. Merino, F. Bolivar, & R. Quintero. 1994. Kinetic study of penicillin acylase production by recombinant E. coli in batch cultures. Process Biochem. 29:197-206. https://doi.org/10.1016/0032-9592(94)85004-6
Raveendran, S., B. Parameswaran, S. B. Ummalyma, A. Abraham, A. K. Mathew, A. Madhavan, S. Rebello, & A. Pandey. 2018. Applications of microbial enzymesin food industry. Food Technol. Biotechnol. 56:16-30. https://doi.org/10.17113/ftb.56.01.18.5491
Razali, R., C. Budiman, K. A. Kamaruzaman, & V. K. Subbiah. 2021. Soluble expression and catalytic properties of codon-optimized recombinant bromelain from MD2 pineapple in Escherichia coli. Protein J. 40:406-418. https://doi.org/10.1007/s10930-021-09974-9
Rehan, F., N. Ahemada, & M. Gupta. 2019. Casein nanomicelle as an emerging biomaterial-A comprehensive review. Colloids Surf. B: Biointerfaces. 179:280-292. https://doi.org/10.1016/j.colsurfb.2019.03.051
Remmert, M., A. Biegert, A. Hauser, & J. Soding. 2011. HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment. Nat. Methods. 9:173-175. https://doi.org/10.1038/nmeth.1818
Roeters, S. J., A. Iyer, G. Pletikapić, V. Kogan, V. Subramaniam, & S. Woutersen. 2017. Evidence for intramolecular antiparallel beta-sheet structure in alpha-synuclein fibrils from a combination of two-dimensional infrared spectroscopy and atomic force microscopy. Sci. Rep. 7:4105. https://doi.org/10.1038/srep41051
Rohin, M. A. K., A. Bakar, C. Abdullah, & A. M. Ali. 2012. Antibacterial activity of flesh and peel methanol fractions of red pitaya, white pitaya and papaya on selected food microorganisms. Int. J. Pharm. Pharm. Sci. 4:185-190.
Rosano, G. L., & E. A. Ceccarelli. 2014. Recombinant protein expression in Escherichia coli: advances and challenges. Front. Microbiol. 5:172. https://doi.org/10.3389/fmicb.2014.00172
Shin, W. H., X. Kang, J. Zhang, & D. Kihara. 2017. Prediction of local quality of protein structure models considering spatial neighbors in graphical models. Sci. Rep. 7:40629. https://doi.org/10.1038/srep40629
Singh, R., A. Mittal, M. Kumar, & P. K. Mehta. 2016. Microbial proteases in commercial applications. J. Pharm. Chem. Biol. Sci. 4:365-374.
Singh, R., M. Kumar, A. Mittal, & P. K. Mehta. 2016. Microbial enzymes: Industrial progress in 21st century. 3 Biotech 6:174. https://doi.org/10.1007/s13205-016-0485-8
Sulthoniyah, S. T. M., Hardoko, & H. Nursyam. 2015. Characterization of extracellular protease lactic acid bacteria from shrimp paste. J. Life Sci. Biomed. 5:01-05.
Waterhouse, A., M. Bertoni, S. Bienert, G. Studer, G. Tauriello, R. Gumienny, F. T. Heer, T.A.P. de Beer, C. Rempfer, L. Bordoli, R. Lepore, & T. Schwede. 2018. SWISS- MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46:W296-W303. https://doi.org/10.1093/nar/gky427
Watson, R. J., I. Schildkraut, B. Q. Qiang, S. M. Martin, & L. P. Visentin. 1982. NdeI: A restriction endonuclease from Neisseria denitrificans which cleaves DNA at 5’-CATATG-3’ sequences. Febs letters. 150:114-116. https://doi.org/10.1016/0014-5793(82)81315-X
Yaraguppi, D. A., B. B. Udapudi, L. R. Patil, V. S. Hombalimath, & A. R. Shet. 2012. In-silico analysis for predicting protein ligand interaction for snake venom protein. J. Adv. Bioinforma. Appl. Res. 3:345-356.
Authors
YusufM., BudimanC., AriefI. I., & SumantriC. (2021). Synthetic Gene-Based Heterologous Expression, Proteolytic, and Structural Characterization of Caseinolytic Protease of Lactobacillus plantarum IIA-1A5. Tropical Animal Science Journal, 44(4), 520-530. https://doi.org/10.5398/tasj.2021.44.4.520
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