Glucose-dependent secretory expression of GFPuv from recombinant yeast cells as a model for a protein-based drug delivery system.

摘要

Encapsulation of live, engineered cells within non-biodegradable or biodegradable, semi-permeable membranes, secreting a therapeutic protein provides a novel drug delivery system. The cells survive via free exchange of nutrients from the surrounding tissue while the therapeutic protein will exit the capsule via simple diffusion. Implantation of such encapsulated systems within the host will, hypothetically, result in the continuous, bioresponsive delivery of the therapeutic proteins at optimal concentrations. The concentrations of the therapeutic peptide may be varied by cloning the responsible gene under the control of a specialized and tightly controlled promoter. This promoter should respond to appropriate signals, in this case, concentrations of glucose. The Green Fluorescent Protein variant, GFPuv, was used as the model protein in this study and secretary expression was established in Saccaharomyces cerevisiae, which constituted the model cell. A plasmid-based vector system was constructed for the secretary expression of GFPuv in yeast. For optimization of methodology, expression from the ADH 2 promoter was characterized using fluorescence microscopy, native dot blots and western blots. Expression was made glucose-dependent by expressing the recombinant cassette from the regulatory regions of the yeast HXT1 and CLN3 genes. Both of these genes are up-regulated in the presence of glucose. Secretory expression from these promoters was further characterized by western blotting and native dot blots.;Glucose-dependent secretary expression of GFPuv thus provides a model which can be used for the therapeutic purpose of insulin delivery to patients suffering from Type I or II diabetes.