Recombinant forms of Gerbich blood group antigens: expression and purification

• Published in: Transfusion clinique et biologique : journal de la Société française de transfusion sanguine Vol. 9; no. 2; pp. 121 - 129
• Main Authors: Jaskiewicz, E, Czerwinski, M, Colin, Y, Lisowska, E
• Format: Journal Article
• Language: English
• Published: Paris Elsevier SAS 01-04-2002; Elsevier
• Subjects: Amino Acid Sequence; Animals; Antibodies, Monoclonal - immunology; antigènes Gerbich; antigènes recombinants; Biological and medical sciences; Blood Group Antigens - biosynthesis; Blood Group Antigens - genetics; Blood Group Antigens - immunology; Blood Group Antigens - isolation & purification; Blood Protein Electrophoresis; Cercopithecus aethiops; CHO Cells; COS Cells; Cricetinae; Cricetulus; Electrophoresis, Polyacrylamide Gel; Exons - genetics; Fundamental and applied biological sciences. Psychology; Gerbich blood group antigens; Glycophorin - biosynthesis; Glycophorin - genetics; Glycophorin - immunology; Glycophorin - isolation & purification; glycophorin C; glycophorine C; Humans; Isoantibodies - immunology; Isoantigens - biosynthesis; Isoantigens - genetics; Isoantigens - immunology; Isoantigens - isolation & purification; Molecular Sequence Data; mutants Yussef et Gerbich; Mutation; recombinant antigens; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - isolation & purification; Sequence Deletion; Structure-Activity Relationship; Transfection; Vertebrates: blood, hematopoietic organs, reticuloendothelial system; Yus and Ge deletion mutants; Gerbich blood group antigens; glycophorin C; antigènes Gerbich; antigènes recombinants; recombinant antigens; Yus and Ge deletion mutants; glycophorine C; mutants Yussef et Gerbich; Flow cytometry; Blood group; Specificity; Glycophorine; Glycoprotein; Glycosylation; Serology; Monoclonal antibody; Mutation; Conveyor; Gerbich system
• ISSN: 1246-7820
• DOI: 10.1016/S1246-7820(02)00233-1

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.
Les formes recombinantes de la glycophorine C (GPC) portant les antigènes de groupe sanguin Gerbich de fréquence élevée et ses mutants naturels de type Yus et Ge (tous porteurs dˈun tag polyhistidine) ont été exprimés dans les cellules CHO et COS 7. Lˈexpression stable de toutes les formes recombinantes de la GPC a été obtenue dans les cellules CHO, mais le niveau dˈexpression est faible et détectable seulement par la cytométrie en flux. Lˈexpression transitoire élevée des formes recombinantes de GPC dans les cellules COS 7 a permis leur purification sur gel dˈagarose Ni–NTA. Les formes recombinantes purifiées de la GPC et de ses mutants de type Yus et Ge se comportent en gel de polyacrylamide–SDS (SDS-PAGE) de façon similaire aux formes natives présentes dans les membranes érythrocytaires. Les mutants GPC.Yus et GPC.Ge apparaissent sous forme de bandes diffuses, suggérant une hétérogénéité de glycosylation identique à celle observée sur les glycoprotéines GPC.Yus et GPC.Ge naturelles. Lˈanalyse par cytométrie en flux des cellules CHO et COS 7 transfectées a montré que la liaison des variants des anticorps monoclonaux anti-GPC était conforme à la spécificité fine de ces anticorps. Les formes recombinantes de GPC transporteurs des antigènes Gerbich communs peuvent être utiles en sérologie, ainsi quˈen tant que modèles pour des études de structure–fonction.