bull. biotechnol. Bulletin of Biotechnology 2717-8323 Avrasya Araştırma Geliştirme Bilim ve Teknoloji Merkezi Limited Şirketi Engineering Mühendislik Investigation of cloning strategies for the recombinant expression of a putative immune modulator TIR domain protein from probiotic Lactobacillus casei 21/1 Bakar Bahar EGE UNIVERSITY https://orcid.org/0000-0002-0627-733X İçier Seyhan MANISA CELAL BAYAR UNIVERSITY Kaplan Türköz Burcu EGE UNIVERSITY 06 19 2020 1 1 23 29 05 10 2020 06 17 2020 Copyright © 2020, Bulletin of Biotechnology 2020 Bulletin of Biotechnology

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.

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