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Single domain affinity proteins for the detection of the genome organizer protein SATB1
KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
KTH, School of Biotechnology (BIO). KTH, Centres, Science for Life Laboratory, SciLifeLab.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Affibody molecules and VHH antibody fragments are two classes of affinity proteins, both characterized by a small size and a single subunit domain structure. Here, we report the selection and characterization of affinity proteins from both classes against the special AT rich sequence binding protein SATB1, a suggested marker protein for aggressive and metastasizing breast cancer. The selected VHH antibody fragments originate from a nonimmune phage display library, while affibody molecules were selected from a library constructed in vitro and displayed on ribosomes. It was observed that selected molecules recognizing one of three conserved DNA-binding domains of SATB1, also recognized its close homologue SATB2 while several of the selected molecules from both classes binding to other regions selectively recognized SATB1. Two of these SATB1 selective molecules, VHH clone 2D2 and affibody molecule clone ZSATB1:2, performed well in differentimmunotechnology applications including ELISA, WB, IF and pull-out experiments and gave a selective staining of endogenous SATB1 in Jurkat T cells. These molecules may thus become useful tools, either alone or in combination, for the selective detection of SATB1 in breast tumor specimens. Due to their small size in comparison to immunoglobulins, such single domain binding proteins may be suitable for high resolution microscopy techniques such as Stimulated Emission Depletion (STED) microscopy, where the resolution may get constrained by the size of the affinity reagent.

Keyword [en]
affibody molecule, VHH antibody fragment, phage display, ribosome display, SATB1
URN: urn:nbn:se:kth:diva-33425OAI: diva2:415413
EU, FP7, Seventh Framework Programme, FLUODIAMON 201 837
QS 2011Available from: 2011-05-06 Created: 2011-05-06 Last updated: 2011-05-06Bibliographically approved
In thesis
1. Ribosome display for selection and evolution of affibody molecules
Open this publication in new window or tab >>Ribosome display for selection and evolution of affibody molecules
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Affinity proteins are invaluable tools in biotechnological and medical applications. This thesis is about combinatorial protein engineering principles for the generation of novel affinity proteins to purify mouse immunoglobulin, detect a potential cancer marker protein or inhibit a cell proliferation pathway.

In a first study, ribosome display was for the first time applied to the selection of so-called affibody molecules, including the design of a ribosome display gene cassette, initial test enrichment experiments and the selection of binders against murine IgG1. One of the selected binders (ZMAB25) showed a highly selective binding profile to murine IgG1, which was exploited in the recovery of two different mouse monoclonal IgG1 antibodies from a bovine immunoglobulin-containing background. Ribosome display was further applied to the selection of affibody molecules binding to SATB1, a suggested marker protein for metastasizing adenocarcinoma. The study also included the selection of VHH antibody fragments from a naïve gene repertoire displayed on phage. Binders from both classes of protein scaffolds could be isolated that selectively recognized SATB1 but not its close homologue SATB2, and were used to detect endogenous SATB1 in Jurkat cells by immunofluorescence microscopy. The well-established phage display technology was used to select affibody molecules binding to H-Ras and Raf-1, both involved in the mitogen-activated protein kinase (MAPK) pathway and playing a central role in the control of cell proliferation, survival and differentiation. An isolated affibody molecule denoted ZRAF322 was found to selectively bind to Raf-1 and inhibit the interaction between H-Ras and Raf-1 in vitro. In a continued effort, ribosome display was applied to the affinity maturation of the ZRAF322 variant in a novel approach, based on repetitive cycles of diversification by error-prone PCR of the entire affibody gene and ribosome display selection, mimicking the principles of natural evolution. The method involved a monitoring of the progress of evolution and variants of ZRAF322 with 13- to 26-fold improved affinities were obtained, that contained different combinations of single or double amino acid substitutions in either previously randomized or framework positions. Implications of the substitutions for binder stability and selectivity were also investigated, showing that a higher affinity could be associated with a lower thermal melting point and that affinity-improved variants showed uncompromised binding selectivity to the hRaf-1 target.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011. xii, 99 p.
Trita-BIO-Report, ISSN 1654-2312 ; 2011:9
affibody, ribosome display, phage display, combinatorial protein engineering, library, murine IgG1, SATB1, Ras, Raf
National Category
Other Industrial Biotechnology
Research subject
SRA - Molecular Bioscience
urn:nbn:se:kth:diva-33191 (URN)978-91-7415-952-3 (ISBN)
Public defence
2011-05-27, Fr4, AlbaNova University Center, Roslagstullsbacken 21, B3, Stockholm, 10:00 (English)
QC 20110506Available from: 2011-05-06 Created: 2011-04-29 Last updated: 2011-05-06Bibliographically approved

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Grimm, SebastianGruselius, JoelNygren, Per-Åke
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