Your browser doesn't support javascript.
loading
Expression, Purification, and Biochemical Characterization of Human Afamin.
Altamirano, Alessandra; Naschberger, Andreas; Fürnrohr, Barbara G; Saldova, Radka; Struwe, Weston B; Jennings, Patrick M; Millán Martín, Silvia; Malic, Suzana; Plangger, Immanuel; Lechner, Stefan; Pisano, Reina; Peretti, Nicole; Linke, Bernd; Aguiar, Mario M; Fresser, Friedrich; Ritsch, Andreas; Lenac Rovis, Tihana; Goode, Christina; Rudd, Pauline M; Scheffzek, Klaus; Rupp, Bernhard; Dieplinger, Hans.
Afiliação
  • Saldova R; NIBRT GlycoScience Group, National Institute for Bioprocessing Research & Training , Dublin, Ireland.
  • Struwe WB; NIBRT GlycoScience Group, National Institute for Bioprocessing Research & Training , Dublin, Ireland.
  • Jennings PM; NIBRT GlycoScience Group, National Institute for Bioprocessing Research & Training , Dublin, Ireland.
  • Millán Martín S; NIBRT GlycoScience Group, National Institute for Bioprocessing Research & Training , Dublin, Ireland.
  • Malic S; Center for Proteomics, Faculty of Medicine, University of Rijeka , 51000 Rijeka, Croatia.
  • Lenac Rovis T; Center for Proteomics, Faculty of Medicine, University of Rijeka , 51000 Rijeka, Croatia.
  • Rudd PM; NIBRT GlycoScience Group, National Institute for Bioprocessing Research & Training , Dublin, Ireland.
  • Dieplinger H; Vitateq Biotechnology GmbH , A-6020 Innsbruck, Austria.
J Proteome Res ; 17(3): 1269-1277, 2018 03 02.
Article em En | MEDLINE | ID: mdl-29441788
Afamin is an 87 kDa glycoprotein with five predicted N-glycosylation sites. Afamin's glycan abundance contributes to conformational and chemical inhomogeneity presenting great challenges for molecular structure determination. For the purpose of studying the structure of afamin, various forms of recombinantly expressed human afamin (rhAFM) with different glycosylation patterns were thus created. Wild-type rhAFM and various hypoglycosylated forms were expressed in CHO, CHO-Lec1, and HEK293T cells. Fully nonglycosylated rhAFM was obtained by transfection of point-mutated cDNA to delete all N-glycosylation sites of afamin. Wild-type and hypo/nonglycosylated rhAFM were purified from cell culture supernatants by immobilized metal ion affinity and size exclusion chromatography. Glycan analysis of purified proteins demonstrated differences in micro- and macro-heterogeneity of glycosylation enabling the comparison between hypoglycosylated, wild-type rhAFM, and native plasma afamin. Because antibody fragments can work as artificial chaperones by stabilizing the structure of proteins and consequently enhance the chance for successful crystallization, we incubated a Fab fragment of the monoclonal anti-afamin antibody N14 with human afamin and obtained a stoichiometric complex. Subsequent results showed sufficient expression of various partially or nonglycosylated forms of rhAFM in HEK293T and CHO cells and revealed that glycosylation is not necessary for expression and secretion.
Assuntos
Palavras-chave

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Tipo de estudo: Prognostic_studies Limite: Animals / Humans Idioma: En Ano de publicação: 2018 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Tipo de estudo: Prognostic_studies Limite: Animals / Humans Idioma: En Ano de publicação: 2018 Tipo de documento: Article