EXPRESSION OF THE MEMBRANE-PROTEIN GLYCOPHORIN-A AS A FUSION WITH THEANTIBIOTIC-RESISTANCE PROTEIN NEOMYCIN PHOSPHOTRANSFERASE-II

The gene for the integral membrane protein glycophorin A (GPA) was clo ned in frame to the 5' end of the antibiotic resistance gene, neomycin phosphotransferase II (NPT). Protein expression was achieved in Esche richia coil as well as in mammalian cells. In case of Chinese hamster ovary cells (CHO) the resistant populations were analyzed 2 weeks afte r transfection; the amount of GPA-NPT fusion protein produced was cons tant from experiment to experiment, Neomycin resistance was directly c orrelated with GPA expression, thus allowing the direct selection for a stable GPA-expressing cell population without the need of a cloning step. The amount of GPA-NPT produced was further increased by weakenin g the specific NPT enzymatic activity via site-directed mutagenesis. D efection was simplified by the fact that all different fusion proteins could be detected by the same anti-NPT antibody. This approach may be also applicable to other membrane proteins. (C) 1998 John Wiley & Son s, Inc.