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.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:NSHD.ca#10222/22284 |
| Date | 30 January 2012 |
| Creators | Livingstone, Scott |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
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