Part:BBa_K3003001

PelB-PenetraMax-SCI57-Rajpal 15 Linker -Ag43

Single Chain Insulin 57 (SCI-57) with Ag43 Cassette can be used for expressing this protein and displaying on the surface of bacteria. We took SCI-57 design from the study “Design of an active ultrastable single-chain insulin analog: synthesis, structure, and therapeutic implications” done by Hua QX et al. SCI-57 differs from other single chain insulin analogs by modification in isoelectric points of determined amino acids and it was aimed for isoelectric points to be near as insulin.Linker peptide provides SCI-57 to be expressed in E. coli as a single chain form. Linker we have used between SCI57's b and a chain was Rajpal 15. This linker is taken from the study “Single-Chain Insulins as Receptor Agonists” done by Rajpal G. et al. This linker provides bioactivity and affinity for single chain insulin to recognized by the receptor, therefore it increases the affinity of SCI for binding to insulin receptor. This composite part is used for SCI-57 protein to be produced in E. Coli and displayed on the surface with Ag43 Cassette and pelB sequence which carries this Ag43 cassette with SCI57 to the membrane. PenetraMax is used for penetration of the cells for insulin to get through the intestinal epithelium into the bloodstream. This peptide is taken from the study “One-month subchronic toxicity study of cell-penetrating peptides for insulin nasal delivery in rats” done by Khafagy et. al. This coding sequence can be used for insulin expression in the surface display than can be cleaved by TEV protease enzyme which recognition site is found in the sequence. TEV Protease is an enzyme that is produced by Tobacco Etch Virus (TEV) and recognition site contained in the sequence is specific for TEV Protease to recognize and cut. Expressed insulin in the surface can be cut by this enzyme and released to the extracellular environment. Also, expressed insulin can be released by Heat Release method. By using this method, insulin will be released from the membrane with the Alpha Subunit of Ag43 as attached form. To isolate insulin and detach it from Alpha Subunit, TEV enzyme can be used.