Customization of recombinant BabA-Lewis b adherence domain as a novel gastric-targeted drug delivery system

Introduction: Lectin-mediated cytoadhesion is a promising targeting concept for gastric drug delivery. Helicobacter pylori, a natural inhabitant of the human stomach, inspired us to use its gastric-adhesion proteins as a novel... [ view full abstract ]

Introduction: Lectin-mediated cytoadhesion is a promising targeting concept for gastric drug delivery. Helicobacter pylori, a natural inhabitant of the human stomach, inspired us to use its gastric-adhesion proteins as a novel gastro-retention drug delivery platform. Our recently published co-crystal structure of H. pylori BabA (Blood group Antigen binding Adhesin), with its physiological ligand Lewis b (Le^b), revealed the existence of a distinct binding domain, the BabA-Crown. However, gastric degradation by pepsin is a major obstacle to any protein-based gastric delivery system. As the ligand-binding region of BabA appears to offer partial resistance to pepsin degradation, we assessed the feasibility of using only the BabA crown for particle targeting.

Methods: The BabA-Crown DNA sequence was amplified by PCR and cloned into pOPE101 periplasmic expression vector. An AviTag sequence was added by site-directed mutagenesis to enable enzymatic monobiotinylation with BirA and subsequent nano/microparticle binding. The resulting constructs were transformed into chemically competent E.coli XL-10 Gold cells. After extraction from bacterial periplasmic space, rBabA-Crown protein was used for assessment of cellular and molecular binding interactions, resistance to pepsin digestion using simulated gastric juice, and microparticle coating.

Results: Our data show binding of the complete extracellular region of BabA (BabA₅₄₇) results in submicron-sized particles able to bind specifically to Le^b. However, BabA₅₄₇ protein was rapidly degraded by pepsin. Attempts to express various truncated forms of rBabA which included the crown region, failed to yield recombinant protein. In contrast, expression of the core binding crown region was successful, but only after addition of chloramphenicol during expression. The resulting protein had partial stability to pepsin digestion. Unexpectedly, circular dichroism data indicated that it may not be folded correctly. Binding to Le^b neoglycoconjugates was revealed at low pH, but this was not specific for Le^b-HSA, as the crown also bound to Le^a-HSA, lacdiNac-HSA and unglycosylated HSA, suggesting non-specific adhesion.

Discussion: Currently, gastric pepsin is a powerful obstacle to protein-based gastric delivery. The use of small, pepsin-resistant polypeptides recognising specific ligands expressed on the gastric epithelium may be a viable approach. However, difficulties with the expression of a correctly folded protein ligand will first need to be overcome.