Recently, it has been discovered that phosphatidyl serine (PS) is exposed on the surface of the vascular endothelium of blood vessels in tumors but not on normal endothelium. Human annexin V, a natural ligand that binds to anionic phospholipids, specifically localizes to vascular endothelium of tumor tissue, but none is detected on endothelium of normal tissue.
The basic idea of this project is that a fusion protein (FP) consisting of L-methioninase linked to annexin V that is injected into the bloodstream will bind to the vascular endothelium in tumors only and will prevent methionine in the bloodstream from reaching the tumor, thereby killing the tumor and/or inhibiting its growth. The use of this FP has the great advantage that it does not have to be delivered directly to the tumor cells but only to the bloodstream, thus overcoming a major disadvantage of protein-based therapeutics for cancer treatment.
A process was developed to produce the FP in Escherichia coli and then purify it. The gene for L-methioninase from Pseudomonas putida was joined to the gene for human annexin V, and the fusion gene was ligated to the pET 30 Ek/LIC vector and transformed into E. coli strain BL21(DE3). This vector adds an amino-terminal 6xHis tag to the overexpressed protein. In the cloning, the gene for the HRV 3C protease was added just ahead of the ATF gene. The FP was overexpressed and then was purified to homogeneity by immobilized metal affinity chromatography. The 6xHis tag was cleaved off using HRV 3C protease. The L-methioninase activity of the FP was measured to be 8.6 U/mg protein.
Data will be presented to demonstrate the binding in vitro of the FP to PS immobilized on plastic microtiter plates and to the surface of human endothelial cells in which PS has been induced to be on the cell surface by the addition of hydrogen peroxide.