Expressão de uma mini proinsulina humana modificada em Pichia pastoris

Abstract

Data by the International Diabetes Foundation (IDF, 2021) demonstrates that diabetes mellitus is a costly epidemic, with 537 million people affected worldwide, 6.7 million deaths each year and with almost 1 trillion dollars spent in treatment around the globe. Due to such worrying metrics, the development of new types of Insulin is necessary to supply global demand and assure the health and wellbeing of those affected. Analogues of recombinant insulin have shown to be efficient and popular alternatives to more traditional variant forms of Insulin, providing flexible and custom treatment to each patient, adapted to the individual needs of the patient. In relation to this, this works has as an objective the production of a proinsulin with a mini C peptide chain and a derivative insulin which combines the characteristics shown by the LisPro modification and a long acting effect provided by the modifications shown in insulin glargine . This work started with the In silico design of the protein, it’s testing and verification through bioinformatics softwares then with its optimization for expression in Pichia pastoris followed by its chemical synthesis. The gene was cloned under the control of the AOX promoter on the expression vector pPIC9, which was initially transformed in Escherichia coli STBL3 to amplify the plasmid DNA for it’s cloning, digestion and integration in the genome of Pichia pastoris GS115. The production of the modified miniproinsulin was done through the induction of BMMY media by 1% methanol at 24 hours intervals in erlenmeyers at 30°C, 180 RPM. The analysis of the protein was done via an tris-tricine SDS-PAGE gel, it’s purification done through IMAC chromatography and immunology validation was done through anti insulin/proinsulin specific antibody via dot-blotting and ELISA. The results show that, as predicted in silico, the modified miniproinsulin has a 6.8KDa molecular mass and an immune response in anti insulin/proinsulin antibodies. This work shows a modified proinsulin analogue with a physio-chemical profile matching in silico predictions, that in future works, will have its physio-chemical properties and physiological response analyzed after conversion to a modified insulin analogue.