Purification of Band 3 from the Human Erythrocyte Membrane and its Incorporation into Liposome

Band 3, the predominent 95,000 dalton anion transport protein, is the major intrinsic glycoprotein of the human erythrocyte membrane. This anion carrier exists as a dimer and binds the cytoskeletons such as spectrin, ankyrin and actin. And the liposomes are vesicular structures which form spontaneouly upon hydration of phospholipids. These artificial lipid vesicles have been investigated as model of the biological membranes and as a mean of improving the delivery of nucleic acids, drugs, proteins and biological substances to specific target tissues and cells. In this study, we were purified Band 3 from the human erythrocyte membrane(ghost) was prepared by hemolysis of intact human erythrocyte with weak alkali-hypotonic solution. Band 6 was removed from ghost by extracting with solution of an ionic strength of 0.15. Band 3 and Band 4 were solubilized selectively by extracting Band 6-depleted ghosts with Triton X-100 under nondenaturing conditions. Band 3 was then purified from Triton X-100 extract treated with p-chloromercuribenzoate by sucrose density gradient ultracentrifugation. This purified Band 3 was incorporated into liposomes prepared by reverse-phase evaporation. Phosphatidyl L-serine and cholesterol(1 : 1 molar ratio) were dissolved in chloroform and then chloroform was removed by rotatory evaporation under reduced pressure. Band 3 solution without Triton X-100 was introduced into a mixture of lipids and diethylether. Diethylether was subsequently removed by evaporation. This purified Band 3 and its incorporation into liposomes were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.