title: Recombinant forms of Gerbich blood group antigens: expression and purification.
abstract: Recombinant forms of normal glycophorin C (GPC), carrying the high frequency Gerbich blood group antigens, and its natural deletion mutants of Yus and Ge type (all combined with oligohistidyl tag) were expressed in CHO and COS 7 cells. The stable expression of all recombinant forms of GPC in CHO cells was obtained, but the level of expression was low and detectable only by flow cytometry. The high level of transient expression of GPC recombinant forms in COS 7 cells allowed their purification on Ni-NTA-agarose. The purified recombinant GPC and mutants of Yus and Ge type behaved in SDS-PAGE similarly to normal GPC forms from RBC membranes. The recombinant GPC.Yus and GPC.Ge mutants appeared as diffuse bands, suggesting the similar heterogeneity of glycosylation that was observed in natural GPC.Yus and GPC.Ge glycoproteins. The flow cytometry analysis of the transfected CHO and COS 7 cells showed that binding of anti-GPC monoclonal antibodies to GPC variants was accordant with the known fine specificity of these antibodies. The obtained recombinant forms of GPC carrying common Gerbich antigens may be useful in serology, and also as model molecules for structure-function studies.
authors.affiliation: Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wroclaw, Poland. jaskiew@immuno.iitd.pan.wroc.pl
authors.forename: E
authors.initials: E
authors.lastname: Jaskiewicz
authors.forename: M
authors.initials: M
authors.lastname: Czerwinski
authors.forename: Y
authors.initials: Y
authors.lastname: Colin
authors.forename: E
authors.initials: E
authors.lastname: Lisowska
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001789:Blood Group Antigens / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D001797:Blood Protein Electrophoresis; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons / Q000235:genetics; D006021:Glycophorins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D007518:Isoantibodies / Q000276:immunology; D007519:Isoantigens / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D001789:Blood Group Antigens; D006021:Glycophorins; D007518:Isoantibodies; D007519:Isoantigens; D011993:Recombinant Fusion Proteins