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Development of affinity columns for the removal of high-abundance proteins in cerebrospinal fluid
KTH, School of Biotechnology (BIO), Proteomics.
Analytical Chemistry, Department of Physical and Analytical Chemistry, Uppsala University.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
Analytical Chemistry, Department of Physical and Analytical Chemistry, Uppsala University.
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2009 (English)In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 52, no 2, 159-166 p.Article in journal (Refereed) Published
Abstract [en]

Various approaches for removal of high-abundance components in body fluids are currently available. While most methods are constructed for plasma depletion, there is a need for body-fluid-specific strategies. The aim of the present study was to design an affinity matrix suitable for the depletion of high-abundance proteins in CSIF (cerebrospinal fluid). Hence, molecules with specific affinity towards proteins present at high concentration in CSIF were desired. Affibody(R) molecules are specific binders of small size that have shown high stability under various conditions and are therefore good candidates for such a matrix. The protein composition in CSF resembles that in plasma. However, 20 % of the proteins are brain-derived and are therefore present in higher proportions in CSF than in plasma, whereas larger plasma-derived proteins are less abundant in CSF. Therefore five high-abundance CSIF proteins were chosen for the design of a CSF-specific depletion setup. Affibody(R) molecules with specificity towards HSA (human serum albumin), IgG, transferrin and transthyretin were combined in an affinity column. In addition, polyclonal antibodies against cystatin C were coupled to chromatographic beads and packed in a separate column. Highly reproducible and efficient removal of the five target proteins was observed. The proportion of depleted proteins were estimated to be 99, 95, 74, 92 and 83 % for HSA, IgG, transferrin, transthyretin and cystatin C respectively. SDS/PAGE analysis was used for monitoring and identifying proteins in native CSF, depleted CSIF samples and the captured fractions. Moreover, shotgun proteomics was used for protein identification in native as well as depleted: CSIF and the achieved data were compared. Enhanced identification of lower abundance components was observed in the depleted fraction, in terms of more detected peptides per protein.

Place, publisher, year, edition, pages
2009. Vol. 52, no 2, 159-166 p.
Keyword [en]
Affibody (R) molecule; affinity resin; biomarker; cerebrospinal fluid (CSF); MS; proteomics
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-8126DOI: 10.1042/BA20080028ISI: 000263202500008Scopus ID: 2-s2.0-60549084220OAI: oai:DiVA.org:kth-8126DiVA: diva2:13364
Note
QC 20100728. Uppdaterad från manuskript till artikel (20100728). Tidigare titel: Development of affinity columns for the removal of high-abundant proteins in cerebrospinal fluidAvailable from: 2008-03-19 Created: 2008-03-19 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Affibody molecules for proteomic and therapeutic applications
Open this publication in new window or tab >>Affibody molecules for proteomic and therapeutic applications
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
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.

Place, publisher, year, edition, pages
Stockholm: KTH, 2008. ix, 73 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2008:3
Keyword
Affibody, protein engineering, phage display, amyloid beta peptide, transferrin, CD25, IL-2 receptor, proteomics
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-4674 (URN)978-91-7178-901-3 (ISBN)
Public defence
2008-04-11, F3, KTH, Lindstedsvägen 26, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100729Available from: 2008-03-19 Created: 2008-03-19 Last updated: 2010-07-29Bibliographically approved

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