Engineering a novel insulin delivery system

Abstract

The studies in this thesis investigated the preparation of a novel system that would be suitable for the oral delivery of insulin. The genetic modification of insulin gene to establish a simple system of insulin production and formulation of polyelectrolyte complex nanoparticles as potential carriers for the delivery of insulin were examined. To facilitate further modifications of insulin gene, several plasmids with insulin were designed and prepared. The design of the plasmids was based on the combination of the simplicity of a bacterial expression system with benefits of the expression in yeast, which is achieved by directing the expressed protein with the signal peptide to the oxidation system of the bacterial periplasmic space. Five plasmids with insulin were successfully prepared (pHB1, pHB2, pHB3, pMD2, pMD3) and all of them expressed insulin, however its amount and form was different. Optimisation of expression conditions was further investigated by testing two induction systems: isopropyl (3-D-1-thiogalactopyranoside (IPTG) and auto-induction and also various temperatures. The impact of those factors on the solubility and localisation of expressed protein was also examined. Expression of the protein was controlled by measuring the optical density of the culture at 600 nm. Samples prepared for expression, solubility and periplasmic localisation test were tested with SDS-PAGE and Western blot techniques. Two plasmids: pMD2 (protein expressed in the periplasm) and pHB1 (the greatest amount of protein expressed in the insoluble form) were selected for further purification process. Final optimisation of the purification protocols was established for selected plasmids. It involved affinity chromatography (Ni column for purification of His tagged proteins) and Achromobacter lyticus protease (ALP) cleavage for purification of periplasmic fraction of protein expressed by plasmid pMD2. A more complex purification process had to be applied for purification of insoluble form of protein expressed by pHB1 plasmid, as the protein had to be isolated, washed, cleaved, purified and finally renaturated. Moreover, optimisation of the refolding reaction was performed using four various redox conditions: glutathione/glutathione disulfide, (3-mercaptoethanol, dithiothreitol and cysteine/cystine, as this reaction was one of the steps that resulted in final low yield of the purification process. The amount of the protein was quantified by BioRad Protein Assay. Additionally samples were analysed with SDS-PAGE and Western blot technique. Prepared plasmids can be used for further genetic modifications as preparing fusions of insulin gene with cell penetrating peptides.