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Affibody-mediated transferrin depletion for proteomics applications
KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi. KTH, Skolan för bioteknologi (BIO), Proteomik.
Affibody AB, Bromma.
KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi. KTH, Skolan för bioteknologi (BIO), Proteomik.
Affibody AB, Bromma.
Vise andre og tillknytning
2007 (engelsk)Inngår i: Biotechnology Journal, ISSN 1860-6768, Vol. 2, nr 11, s. 1389-1398Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

An Affibody® (Affibody) ligand with specific binding to human transferrin was selected by phage display technology from a combinatorial protein library based on the staphylococcal protein A (SpA)-derived Z domain. Strong and selective binding of the selected Affibody ligand to transferrin was demonstrated using biosensor technology and dot blot analysis. Impressive specificity was demonstrated as transferrin was the only protein recovered by affinity chromatography from human plasma. Efficient Affibody-mediated capture of transferrin, combined with IgG- and HSA-depletion, was demonstrated for human plasma and cerebrospinal fluid (CSF). For plasma, 85% of the total transferrin content in the samples was depleted after only two cycles of transferrin removal, and for CSF, 78% efficiency was obtained in single-step depletion. These results clearly suggest a potential for the development of Affibody-based resins for the removal of abundant proteins in proteomics analyses.

sted, utgiver, år, opplag, sider
2007. Vol. 2, nr 11, s. 1389-1398
Emneord [en]
Affibody ligand; Affinity resin; Protein engineering; Proteomics; Transferrin
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-8125DOI: 10.1002/biot.200700053OAI: oai:DiVA.org:kth-8125DiVA, id: diva2:13363
Merknad
QC 20100728Tilgjengelig fra: 2008-03-19 Laget: 2008-03-19 Sist oppdatert: 2010-07-28bibliografisk kontrollert
Inngår i avhandling
1. Affibody molecules for proteomic and therapeutic applications
Åpne denne publikasjonen i ny fane eller vindu >>Affibody molecules for proteomic and therapeutic applications
2008 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

This thesis describes generation and characterization of Affibody molecules with future applications in proteomics research, protein structure determinations, therapeutic treatment of disease and medical imaging for in vivo diagnostics. Affibody molecules are engineered affinity proteins developed by combinatorial protein engineering from the 58-residue protein A-derived Z domain scaffold. Novel Affibody molecules targeting human proteins were selected from a combinatorial library using phage display technology.

In the first two investigations, an Affibody molecule specifically targeting the high abundant human serum protein transferrin was generated. The intended future use of this Affibody ligand would be as capture ligand for depletion of transferrin from human samples in proteomics analysis. Strong and highly specific transferrin binding of the selected Affibody molecule was demonstrated by biosensor technology, dot blot analysis and affinity chromatography. Efficient Affibody-mediated depletion of transferrin in human plasma and cerebrospinal fluid (CSF) was demonstrated in combination with IgG and HSA removal. Furthermore, depletion of five high abundant proteins including transferrin from human CSF gave enhanced identification of proteins in a shotgun proteomics analysis.

Two studies involved the selection and characterization of Affibody molecules recognizing Alzheimer’s amyloid beta (Abeta) peptides. Future prospect for the affinity ligands would primarily be for therapeutic applications in treatment of Alzheimer’s disease. The developed A-binding Affibody molecules were found to specifically bind to non-aggregated forms of Abeta and to be capable of efficiently and selectively capture Abeta peptides from spiked human serum. Interestingly, the Abeta-binding Affibody ligands were found to bind much better to Abeta as dimeric constructs, and with impressive affinity as cysteine-bridged dimers (KD~17 nM). NMR spectroscopy studies revealed that the original helix one, of the two Affibody molecules moieties of the cysteine-bridged dimers, was unfolded upon binding, forming intermolecular β-sheets that stabilized the Abeta peptide, enabling a high resolution structure of the peptide. Furthermore, the Abeta-binding Affibody molecules were found to inhibit Abeta fibrillation in vitro.

In the last study, Affibody molecules directed to the interleukin 2 (IL-2) receptor alpha (CD25) were generated. CD25-binding Affibody molecules could potentially have a future use in medical imaging of inflammation, and possibly in therapeutic treatment of disease conditions with CD25 overexpression. The selected Affibody molecules were demonstrated to bind specifically to human CD25 with an apparent affinity of 130-240 nM. Moreover, the CD25-targeting Affibody molecules were found to have overlapping binding sites with the natural ligand IL-2 and an IL-2 blocking monoclonal antibody. Furthermore, the Affibody molecules demonstrated selective binding to CD25 expressing cells.

sted, utgiver, år, opplag, sider
Stockholm: KTH, 2008. s. ix, 73
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2008:3
Emneord
Affibody, protein engineering, phage display, amyloid beta peptide, transferrin, CD25, IL-2 receptor, proteomics
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-4674 (URN)978-91-7178-901-3 (ISBN)
Disputas
2008-04-11, F3, KTH, Lindstedsvägen 26, Stockholm, 10:00
Opponent
Veileder
Merknad
QC 20100729Tilgjengelig fra: 2008-03-19 Laget: 2008-03-19 Sist oppdatert: 2010-07-29bibliografisk kontrollert

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