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Pregled bibliografske jedinice broj: 1049761

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 et al.
Expression, Purification, and Biochemical Characterization of Human Afamin // Journal of Proteome Research, 17 (2018), 3; 1269-1277 doi:10.1021/acs.jproteome.7b00867 (međunarodna recenzija, članak, znanstveni)

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Naslov
Expression, Purification, and Biochemical Characterization of Human Afamin

Autori
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

Izvornik
Journal of Proteome Research (1535-3893) 17 (2018), 3; 1269-1277

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
afamin ; expression in different cellular models ; glycosylation heterogeneity

Sažetak
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

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti



POVEZANOST RADA

Ustanove:
Medicinski fakultet, Rijeka

Profili:

Avatar Url Tihana Lenac Roviš (autor)

Poveznice na cjeloviti tekst rada:

doi pubs.acs.org

Citiraj ovu publikaciju:

Altamirano, Alessandra; Naschberger, Andreas; Fürnrohr, Barbara G.; Saldova, Radka; Struwe, Weston B.; Jennings, Patrick M.; Millán Martín, Silvia; Malic, Suzana; Plangger, Immanuel; Lechner, Stefan et al.
Expression, Purification, and Biochemical Characterization of Human Afamin // Journal of Proteome Research, 17 (2018), 3; 1269-1277 doi:10.1021/acs.jproteome.7b00867 (međunarodna recenzija, članak, znanstveni)
Altamirano, A., Naschberger, A., Fürnrohr, B., Saldova, R., Struwe, W., Jennings, P., Millán Martín, S., Malic, S., Plangger, I. & Lechner, S. (2018) Expression, Purification, and Biochemical Characterization of Human Afamin. Journal of Proteome Research, 17 (3), 1269-1277 doi:10.1021/acs.jproteome.7b00867.
@article{article, author = {Altamirano, Alessandra and Naschberger, Andreas and F\"{u}rnrohr, Barbara G. and Saldova, Radka and Struwe, Weston B. and Jennings, Patrick M. and Mill\'{a}n Mart\'{\i}n, Silvia and Malic, Suzana and Plangger, Immanuel and Lechner, Stefan and Pisano, Reina and Peretti, Nicole and Linke, Bernd and Aguiar, Mario M. and Fresser, Friedrich and Ritsch, Andreas and Lenac Rovis, Tihana and Goode, Christina and Rudd, Pauline M. and Scheffzek, Klaus and Rupp, Bernhard and Dieplinger, Hans}, year = {2018}, pages = {1269-1277}, DOI = {10.1021/acs.jproteome.7b00867}, keywords = {afamin, expression in different cellular models, glycosylation heterogeneity}, journal = {Journal of Proteome Research}, doi = {10.1021/acs.jproteome.7b00867}, volume = {17}, number = {3}, issn = {1535-3893}, title = {Expression, Purification, and Biochemical Characterization of Human Afamin}, keyword = {afamin, expression in different cellular models, glycosylation heterogeneity} }
@article{article, author = {Altamirano, Alessandra and Naschberger, Andreas and F\"{u}rnrohr, Barbara G. and Saldova, Radka and Struwe, Weston B. and Jennings, Patrick M. and Mill\'{a}n Mart\'{\i}n, Silvia and Malic, Suzana and Plangger, Immanuel and Lechner, Stefan and Pisano, Reina and Peretti, Nicole and Linke, Bernd and Aguiar, Mario M. and Fresser, Friedrich and Ritsch, Andreas and Lenac Rovis, Tihana and Goode, Christina and Rudd, Pauline M. and Scheffzek, Klaus and Rupp, Bernhard and Dieplinger, Hans}, year = {2018}, pages = {1269-1277}, DOI = {10.1021/acs.jproteome.7b00867}, keywords = {afamin, expression in different cellular models, glycosylation heterogeneity}, journal = {Journal of Proteome Research}, doi = {10.1021/acs.jproteome.7b00867}, volume = {17}, number = {3}, issn = {1535-3893}, title = {Expression, Purification, and Biochemical Characterization of Human Afamin}, keyword = {afamin, expression in different cellular models, glycosylation heterogeneity} }

Časopis indeksira:


  • Current Contents Connect (CCC)
  • Web of Science Core Collection (WoSCC)
    • Science Citation Index Expanded (SCI-EXP)
    • SCI-EXP, SSCI i/ili A&HCI
  • Scopus
  • MEDLINE

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