Expression, Purification, and in Vivo Evaluation of GFP-Fused M Cell Targeting Receptor Binding Domain of Protein FIMH

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Author(s): Thanh-Hoa Nguyen-Thi, Kien-Quang Huynh, Phuong-Linh Dinh-Thi, Linh-Thuoc Tran, Yong Suk Jang, Hieu Tran-Van*.

Journal Name: Protein & Peptide Letters

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Background: The number of oral vaccines is still limited due to many difficulties suffered in the intestinal environment, such as mucosal clearance, vast area, harsh conditions, deteriorative enzymes, impermeability, tolerance, etc. Numerous strategies have focused on directing antigen to the receptors of M cells, which is the main gateway to acquire and initiate specific responses to antigens in intestine. FimHrb is a receptor binding domain of type 1 of fimbriae from E. coli and Salmonella that can bind to GP2 receptor expressed exclusively on M cells.

Objective: In this study, we evaluated the potential of FimHrb for oral vaccine development via its ability to adhere M to cells.

Methods: The coding gene of FimHrb fused Green Fluorescent Protein (GFP) was cloned and expressed intracellularly in E. coli host strain. The recombinant protein FimHrb-GFP was then purified by IMAC method through 6x His tag designed downstream of GFP. Finally, the purified protein was monitored its binding on murine M cells in Payer Patch region.

Results: Following the methods mentioned above, the coding gene FimHrb-GFP was successfully cloned into vector pET22b and intracellularly expressed in soluble form at low temperature induction. The purity and the recovered yield of this protein were 90% and 20%, respectively. After that, the adhesion of FimHrb-GFP was monitored in murine small intestine, which showed that the protein bound to Peyer Patch region and did not restrict on M cells.

Conclusion: With the present data, we revealed a candidate protein FimHrb targeted receptor on M cells for oral vaccine development and other factors in E. coli would supplement FimH to provide the specific invasion of these bacteria via M cells.

Keywords: E. coli expression, FimH, low temperature induction, M cell, mucosal immunity, oral vaccines

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(E-pub Ahead of Print)
DOI: 10.2174/0929866526666190405122805
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