Part:BBa_K3003021

Single Chain Insulin-57 with a linker peptide (Rajpal 15)

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.

All single transformed IDT BL21 cells were induced with IPTG. Western blot was performed following the heat release. All constructs were fused to Ag43. Therefore we were expecting to see bands around 46 kDa.IDT G3 (Rajpal 15 design with SCI57 a-b chains) and IDT G7 (SCI57 with its own linker a-b chains) were found out to be expressed.

We performed immunocytochemistry (ICC) assay in order to observe the expression of our SCI constructs. Working principle of immunocytochemistry assay is to provide bacterial cultures that are producing His-Tag containing proteins with His-Tag binding antibodies and visualize the cells under fluorescence microscopy. With primary antibodies, we bind the His-Tag that is expressed with the SCI that is secreted to the cell surface. After primary antibodies, secondary antibodies are used in order to label the primary antibodies with fluorescence activity. Under fluorescence microscopy these cells are visualised.