Expression of the membrane protein glycophorin A as a fusion with the antibiotic resistance protein neomycin phosphotransferase II.
The gene for the integral membrane protein glycophorin A (GPA) was cloned in frame to the 5' end of the antibiotic resistance gene, neomycin phosphotransferase II (NPT). Protein expression was achieved in Escherichia coli as well as in mammalian cells. In case of Chinese hamster ovary cells (CHO) the resistant populations were analyzed 2 weeks after transfection; the amount of GPA-NPT fusion protein produced was constant from experiment to experiment. Neomycin resistance was directly correlated 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 weakening the specific NPT enzymatic activity via site-directed mutagenesis. Detection 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.
Krömer
W J
WJ
Institute of Biotechnology, ETH Zürich, CH8093 Zürich, Switzerland; telephone +411 633 31 70.
Bailey
J E
JE
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006021:Glycophorins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D019868:Kanamycin Kinase / Q000096:biosynthesis / Q000235:genetics*; D007613:Kanamycin Resistance / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014170:Transformation, Genetic
D018076:DNA, Complementary; D006021:Glycophorins; D011993:Recombinant Fusion Proteins; D019868:Kanamycin Kinase