Expression, Purification, and Biochemical Characterization of Human Afamin

• Published in: Journal of proteome research 2018-03, Vol.17 (3), p.1269-1277
• Main Authors: 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
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - metabolism; Antigen-Antibody Complex - chemistry; Antigen-Antibody Complex - metabolism; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; CHO Cells; Cloning, Molecular; Cricetulus; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Genetic Vectors - chemistry; Genetic Vectors - metabolism; Glycoproteins - chemistry; Glycoproteins - genetics; Glycoproteins - metabolism; Glycosylation; HEK293 Cells; Humans; Immunoglobulin Fab Fragments - chemistry; Immunoglobulin Fab Fragments - metabolism; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - chemistry; Polysaccharides - chemistry; Polysaccharides - metabolism; Protein Processing, Post-Translational; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Serum Albumin, Human - chemistry; Serum Albumin, Human - genetics; Serum Albumin, Human - metabolism
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/acs.jproteome.7b00867

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.