Part:BBa_K3003003

PelB-Penetramax-SCI57-Rajpal 27 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. The linker we have used between SCI57's b and a chain was Rajpal 27. This linker is taken from the study “Single-Chain Insulins as Receptor Agonists” done by Rajpal G. et al. This linker provides more bioactivity and affinity for single chain insulin to recognized by the receptor rather than Rajpal 15, therefore it increases the affinity of SCI for binding to insulin receptor in a more efficient way. 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 coding sequence can be used for insulin expression in the surface display than can be cleaved by the 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.