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Selection and characterisation of affibody molecules inhibiting the interaction between Ras and Raf in vitro
KTH, School of Biotechnology (BIO), Proteomics.
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0001-7034-0850
KTH, School of Biotechnology (BIO), Proteomics.
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2010 (English)In: NEW BIOTECHNOL, ISSN 1871-6784, Vol. 27, no 6, 766-773 p.Article in journal (Refereed) Published
Abstract [en]

Development of molecules with the ability to selectively inhibit particular protein-protein interactions is important in providing tools for understanding cell biology In this work, we describe efforts to select small Ras- and Raf-specific three-helix bundle affibody binding proteins capable of inhibiting the interaction between H-Ras and Raf-1, from a combinatorial library displayed on bacteriophage Target-specific variants with typically high nanomolar or low micromolar affinities (K-D) could be selected successfully against both proteins, as shown by dot blot, ELISA and real-time biospecific interaction analyses Affibody molecule variants selected against H-Ras were shown to bind epitopes overlapping each other at a site that differed from that at which H-Ras interacts with Raf-1 In contrast, an affibody molecule isolated during selection against Raf-1 was shown to effectively inhibit the interaction between H-Ras and Raf-1 in a dose-dependent manner Possible intracellular applications of the selected affibody molecules are discussed

Place, publisher, year, edition, pages
2010. Vol. 27, no 6, 766-773 p.
National Category
Other Industrial Biotechnology Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-30521ISI: 000285896200008ScopusID: 2-s2.0-78649713667OAI: diva2:401956
Swedish Research Council
QC 20110304Available from: 2011-03-04 Created: 2011-02-28 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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