Generation Of Cell-Penetrating Heme Oxygenase Proteins To Improve The Resistance Of Steatotic Livers To Reperfusion Injury Following Transplantation

Abstract

Liver transplantation is the only life-saving treatment for patients with end-stage liver disease; however, organ availability is insufficient to meet demands. Steatotic livers are extended criteria donor (ECD) organs that could be used for transplantation if not for an increased susceptibility ischemia reperfusion injury (IRI). Heme oxygenase-1 is a gene, that when upregulated has be shown to reduce IRI in animal models of transplantation. Increasing HO-1 activity in steatotic livers by delivery of a functional cell-penetrating HO-1 protein (through the use of cell-penetrating peptides) may provide protection against IRI, making these organs useful for transplantation. The purpose of this thesis was the generation and testing of a cell-penetrating HO-1 protein. HO-1 and EGFP gene sequences were cloned into the pET-28B(+) vector in frame with a CPP or TAT sequence. Resulting plasmids were cloned into E. coli, and protein expression was induced using IPTG. Proteins were purified using Ni-NTA affinity chromatography under denaturing and non-denaturing conditions. Non-denatured proteins were tested for HO-1 activity and the ability of both denatured and non-denatured proteins to transduce cells in vitro was tested by fluorescence microscopy. The cell-penetrating ability of nondenatured proteins was further tested in J774, HepG2 and HUVEC cells using immunofluorescence. Five HO-1 and two EGFP cell-penetrating proteins were generated expressed and purified successfully. Purified non-denatured HO-1 retains its enzymatic activity. Non-denatured CPP-EGFP and CPP-HO1 penetrated cells more effectively than their denatured counterparts. CPP-EGFP and CPP-HO1 proteins are able to penetrate multiple cell types in vitro. Successful generation and testing of a cell-penetrating HO-1 protein, for use in an animal model of steatotic liver transplantation. This protein demonstrates promise for use as a potential therapeutic agent in the field of liver transplantation.