Investigation of cloning strategies for the recombinant expression of a putative immune modulator TIR domain protein from probiotic Lactobacillus casei 21/1

Year 2020, Volume: 1 Issue: 1, 23 - 29, 19.06.2020

Bahar Bakar , Seyhan İçier Burcu Kaplan Türköz

Abstract

TIR domain proteins have a key role in Toll-Like Receptor (TLR) signalling pathway in innate immunity. Bacteria can produce TIR domain proteins and those from pathogens were shown to manipulate TLR signaling via mimicking host proteins. Probiotics can also affect TLR signaling, but the molecular details have not been yet elucidated. In this study, a putative protein from probiotic Lactobacillus casei was identified as a TIR domain protein (LcTIR) based on sequence conservation. Multiple sequence alignments showed that LcTIR has high similarity to known TIR domains and the structural model of LcTIR verified the presence of the TIR domain fold. Following this, the gene encoding LcTIR was cloned in several Escherichia coli plasmids in order to obtain pure protein for structural and biochemical studies. Several fusion partners, promoter systems, different E.coli host strains and induction conditions were investigated to achieve recombinant protein production. In all conditions, recombinant LcTIR was produced at low amounts. The highest amount of protein obtained was GST-LcTIR fusion; in Rosetta(DE3)pLysS cells at 37°C with 0.5mM IPTG induction, where nearly all the protein was found in inclusion bodies. Furthermore, in all the constructs and strains tested, the low amount of LcTIR production suppressed cell growth and this might indicate its potential as an antimicrobial agent which opens a new era on bacterial TIR domains. This study is one of the first studies investigating the presence of probiotic TIR domain proteins, and future studies are needed to obtain soluble protein to assay their effect on TLR signalling mechanisms.

Keywords

TIR domain proteins, molecular cloning, probiotic, structural bioinformatics, toxic recombinant protein

Supporting Institution

Scientific and Technical Research Council of Turkey (TÜBİTAK)

Project Number

Project No: 116Z299

Thanks

This research was supported by the Scientific and Technical Research Council of Turkey, TUBITAK (Project No: 116Z299). Authors would like to thank to EMBL for pETM11-SUMO3GFP plasmid and M.Sc.Dicle Dilara Akpınar for technical assistance.

References

• Ahmad I, Nawaz N, Darwesh NM, ur Rahman S, Mustafa MZ, Khan SB, Patching SG (2018) Overcoming challenges for amplified expression of recombinant proteins using Escherichia coli. Protein Expression Purif 144:12–18. doi:10.1016/j.pep.2017.11.005
• Castillo NA, Perdigán G, De Moreno De Leblanc A (2011) Oral administration of a probiotic Lactobacillus modulates cytokine production and TLR expression improving the immune response against Salmonella enterica serovar Typhimurium infection in mice. BMC Microbiol 11(1):177. doi:10.1186/1471-2180-11-177
• Chaudhar A, Ganguly K, Cabantous S, Waldo GS, Micheva-Viteva SN, Nag K., Hlavacek WS, Tung CS (2012) The Brucella TIR-like protein TcpB interacts with the death domain of MyD88. Biochem Biophys Res Commun 417(1):299–304. doi:10.1016/j.bbrc.2011.11.104
• Chhetri G, Kalita P, Tripathi T (2015) An efficient protocol to enhance recombinant protein expression using ethanol in Escherichia coli. MethodsX 2:385–391. doi:10.1016/j.mex.2015.09.005
• Cirl C, Wieser A, Yadav M, Duerr S, Schubert S, Fischer H, Stappert D, Wantia N, Rodriguez N, Wagner H, Svanborg C, Miethke T (2008) Subversion of Toll-like receptor signaling by a unique family of bacterial Toll/interleukin-1 receptor domain-containing proteins. Nat Med 14(4):399–406. doi.org/10.1038/nm1734
• EMBL (2019) Decreasing Protein Toxicity. Available via Dialog. https://www.embl.de/pepcore/pepcore_services/index.html of subordinate document. Accesed 10 June 2019
• Galdeano CM, de Moreno de LeBlanc A, Vinderola G, Bonet MEB, Perdigon G (2007) Proposed model: mechanisms of immunomodulation induced by probiotic bacteria. Clin Vaccine Immunol 14(5):485–492. doi.org/10.1128/CVI.00406-06
• Hoarau C, Lagaraine C, Martin, L (2006) Supernatant of Bifidobacterium breve induces dendritic cell maturation, activation, and survival through a Toll-like receptor 2 pathway. J Allergy Clin Immunol 50:696–702. doi.org/10.1016/j.jaci.2005.10.043
• Jeon SG, Kayama H, Ueda Y, Takahashi T, Asahara T, Tsuji H, Tsuji NM, Kiyono, H., Ma, J. S. Kusu, T., Okumura, R., Hara, H., Yoshida, H., Yamamoto, M., Nomoto, K. Takeda K. (2012) Probiotic Bifidobacterium breve induces IL-10-producing Tr1 cells in the colon. PLoS Pathog, 8(5):1–15. doi.org/10.1371/journal.ppat.1002714
• Kallberg M, Wang H, Wang S, Peng J, Wang Z, Lu H, Xu J (2016) Template-based protein structure modeling using the RaptorX web server. Nat Protoc 7(8):1511–1522. doi.org/10.1038/nprot.2012.085
• Kaplan-Türköz B (2017) A putative Toll/interleukin-1 receptor domain protein from Helicobacter pylori is dimeric in solution and interacts with human Toll-like receptor adaptor myeloid differentiation primary response 88. Microbiol Immunol 61(2):85–91. doi.org/10.1016/j.febslet.2013.09.007
• Kaplan-Türköz B, Koelblen T, Felix C, Candusso MP, O’Callaghan D, Vergunst AC, Terradot L (2013) Structure of the Toll/interleukin 1 receptor (TIR) domain of the immunosuppressive Brucella effector BtpA/Btp1/TcpB. FEBS Lett 587(21):3412–3416. doi: 10.1016/j.febslet.2013.09.007
• Kaur J, Kumar A, Kaur, J (2018) International Journal of Biological Macromolecules Strategies for optimization of heterologous protein expression in E . coli : Roadblocks and reinforcements. Int J Biol Macromol 106:803–822. doi.org/10.1016/j.ijbiomac.2017.08.080
• Kitazawa H, Watanabe H, Shimosato T, Kawai Y, Itoh T, Saito, T (2003) Immunostimulatory oligonucleotide, CpG-like motif exists in Lactobacillus delbrueckii ssp. bulgaricus NIAI B6. Int J Food Microbiol 85(1):11–21. doi.org/10.1016/S0168-1605(02)00477-4
• Marblestone JG, Edavettal SC, Lim Y, Lim P, Zuo XUN, Butt TR (2006) Comparison of SUMO fusion technology with traditional gene fusion systems : Enhanced expression and solubility with SUMO. Protein Sci 15:182–189. doi.org/10.1110/ps.051812706.for
• Newman RM, Salunkhe P, Godzik A, Reed JC (2006) Identification and characterization of a novel bacterial virulence factor that shares homology with mammalian Toll/interleukin-1 receptor family proteins. Infection Immun, 74(1):594–601. doi.org/10.1128/IAI.74.1.594-601.2006
• Notredame C, Higgins DG, Heringa J (2000) T-coffee: A novel method for fast and accurate multiple sequence alignment. J Mol Biol 302(1):205–217. doi.org/10.1006/jmbi.2000.4042
• O’Neill LAJ, Bowie AG (2007) The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling. Nat Rev Immunol 7(5):353–364. doi.org/10.1038/nri2079
• Radhakrishnan GK, Yu Q, Harms JS, Splitter GA (2009) Brucella TIR domain-containing protein mimics properties of the toll-like receptor adaptor protein TIRAP. J Biol Chem 284(15): 9892–9898. doi.org/10.1074/jbc.M805458200
• Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli : advances and challenges, Front Microbiol 5:1–17. doi.org/10.3389/fmicb.2014.00172
• Salcedo SP, Marchesini MI, Lelouard H, Fugier E, Jolly G, Balor S, Muller A, Lapaque N, Demaria O, Alexopoulou L, Comerci DJ, Ugalde RA, Pierre P, Gorvel JP (2008) Brucella control of dendritic cell maturation is dependent on the TIR-containing protein Btp1. PLoS Pathog 4(2):e21. doi.org/10.1371/journal.ppat.0040021
• Saluta M, Bell PA (1998) Troubleshooting GST fusion protein expression in E.coli. Life Sci News 1:1–3.
• Snyder GA, Cirl C, Jiang J, Chen K, Waldhuber A, Smith PT, Durr S, Cirl C, Jiang J, Jennings W, Luchetti T, Snyder N, Sundberg EJ, Wintrode P, Miethke T, Xiao TS (2013) Molecular mechanisms for the subversion of MyD88 signaling by TcpC from virulent uropathogenic Escherichia coli. Proc Natl Acad Sci U.S.A. 110(17):6985–6990. doi.org/10.1073/pnas.1215770110
• Suter-Crazzolara C, Unsicker K (1995) Improved expression of toxic proteins in E. coli. BioTechniques, 19(2), 202—204.
• ThermoFisher (2019) GST-tagged Proteins – Production and Purification. Available via Dialog. https://www.thermofisher.com/tr/en/home/life-science/protein-biology.html of subordinate document. Accessed 15 March 2019
• Tripathi NK, Shrivastava A (2019) Recent Developments in Bioprocessing of Recombinant Proteins : Expression Hosts and Process Development. Front Bioengi Biotech 7:420. doi.org/10.3389/fbioe.2019.00420
• Turner JD (2003) A bioinformatic approach to the identification of bacterial proteins interacting with Toll-interleukin 1 receptor-resistance (TIR) homology domains. FEMS Immunol Med Mic 37(1):13–21. doi.org/10.1016/S0928-8244(03)00095-6
• Ve T, Williams SJ, Kobe B (2015) Structure and function of Toll/interleukin-1 receptor/resistance protein (TIR) domains. Apoptosis 20(2):250–261. doi.org/10.1007/s10495-014-1064-2
• Wang Y, Xie J, Li Y, Dong S, Liu H, Chen J, Wang Y, Zhao S, Zhang Y, Zhang H (2016) Probiotic Lactobacillus casei Zhang reduces pro-inflammatory cytokine production and hepatic inflammation in a rat model of acute liver failure. Eur J Nutr 55(2):821–831. doi.org/10.1007/s00394-015-0904-3
• Zhang Q, Zmasek CM, Cai X, Godzik A (2012) TIR domain-containing adaptor SARM is a late addition to the ongoing microbe–host dialog. Dev Comp Immunol, 35(4):461–468. doi.org/10.1016/j.dci.2010.11.013