Bio-mimicry of paracellular surface on implant neck surface by immobilizing functional claudin peptide to establish an epithelial seal

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The aim of this research project is to immobilize recombinantly synthesized claudin membrane protein for epithelial cell attachment on an inert titanium surface to achieve soft tissue attachment around dental implant collar. Claudins DNA sequence was amplified from the human cDNA library using claudin specific primers. Subsequently, the amplified DNA sequence was ligated to pET-22 vector and inserted into Escherichia coli (E.coli) bacteria for cloning and transfected to BL-21 bacterial cells for protein synthesis. Claudin protein expression was attempted at several temperatures and variable durations to determine the best case scenario. Protein was usually expressed as inclusion bodies (IBs), and there were challenges despite several attempts using various solubilizing agents to solubilize these IBs. However, such hurdles were previously expected by the researcher since claudins are membrane proteins and consists of several hydrophobic amino acids, causing IB formation.Currently, to overcome these challenges the researcher will use non-bacterial protein synthesis such as cell-free systems for claudin protein synthesis to avoid IB formation and dependency on bacterial cells for protein expression.In the meantime, commercially claudin protein was immobilized on titanium discs with bio-functional groups and linkers for gingival epithelial cells adhesion to surface modified titanium discs. The preliminary tasks for protein expression using cell-free protein expression systems have already begun as of now.