Expression, Purification, and Biochemical Characterization of Human Afamin

Alessandra Altamirano, Andreas Naschberger, Barbara G. Fürnrohr, Radka Saldova, Weston B. Struwe, Patrick M. Jennings, Silvia Millán Martín, Suzana Malic, Immanuel Plangger, Stefan Lechner, Reina Pisano, Nicole Peretti, Bernd Linke, Mario M. Aguiar, Friedrich Fresser, Andreas Ritsch, Tihana Lenac Rovis, Christina Goode, Pauline M. Rudd, Klaus ScheffzekBernhard Rupp, Hans Dieplinger

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

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.
Original languageEnglish (US)
Pages (from-to)1269-1277
Number of pages9
JournalJournal of Proteome Research
Volume17
Issue number3
DOIs
StatePublished - Mar 2 2018
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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