Electroinsertion of xeno proteins in red blood cell membranes yields a long lived protein carrier in circulation

Abstract

Electroinsertion, a novel method of implanting xeno-proteins in red blood cell plasma membranes is applied to the insertion of human glycophorin in mouse red blood cells. The procedures yield erythrocytes with approx. 10 4 glycophorin molecules per cell, displaying the patching phenomenon when reated with antiglycophorin monoclonal antibodies. Insertion of FITC-labeled glycophorin and subsequent quenching of FITC fluorescence with anti FITC antibody indicated that 70% of the inserted molecules were ‘correctly’ oriented, displaying the epitopes shown by glycophorin in human red blood cells. Moreover, insertion of FITC glycophorin in the red blood cell membranes yielded, under the fluorescence microscope continuous fluorescence which became patchy after reaction with anti-glycophorin monoclonal antibodies. When injected in mice, biotinylated RBC-Glyc + were shown to have the same life-span as normal mouse red blood cells, i.e. t 1/2 ≈ 12 days. Cytofluorometric assay of human glycophorin inserted in the red blood cell showed the same life-span for the inserted protein as for the red blood cell, with the protein fully ‘functional’, i.e. capable of binding antibodies. Human glycophorin inserted in mouse erythrocyte did not elicit any immune response in mice, whereas the same concentrations of free glycophorin injected i.v. were highly immunogenic.