Change search
ReferencesLink to record
Permanent link

Direct link
Affinity maturation of an affibody molecule binding to human Raf-1 via non-targeted in vitro evolution
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0003-4214-6991
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The use of in vitro protein library technologies for the generation of high affinity binding proteins often includes an affinity maturation step, involving the construction of secondary libraries from which second generation variants with improved affinities are selected. We describe a novel approach for the affinity maturation of affibody molecules based on stepwise in vitro molecular evolution, involving cycles of whole gene error-prone PCR amplification for the introduction of diversity over the entire 58-residue three-helix bundle structure and ribosome display for selection. Starting with a human Raf-1 binding affibody molecule of an initial 2 μM dissociation constant (KD), the in vitro evolution process was followed on both gene and protein levels via DNA sequencing and a biosensor based monitoring of the collective target binding ability of expressed pools obtained after each selection cycle. After a first cycle of diversification and selection, no increase in the hRaf-1 target binding of the pool was observed, whereas after two cycles, a significant increase in the binding response was seen. DNA sequencing showed that an alanine to valine substitution in an earlier variegated position had been effectively enriched, and was present in 11% and 83% of all clones after cycle one and two, respectively, either alone or in combination with other substitutions. Further studies on a subset of individual variants isolated after cycle two showed that the observed A27V substitution alone accounted for a 13-fold increase in affinity, predominantly through increased on-rate kinetics. Additional substitutions in framework or non-framework positions typically resulted in a further two-fold increase in affinity. Interestingly, thermal melting point (Tm) analyses showed that an increased affinity could be associated with either slightly higher or lower Tm values, compared to the parental variant. All investigated variants showed excellent refolding properties, and the selectivity of the affinity matured hRaf-1 binders had not been compromised by the substitutions, as analyzed using a multiplexed bead-based binding assay involving 77 recombinant human control protein fragments.

Keyword [en]
affibody molecule, ribosome display, error-prone PCR, library, evolution
National Category
Industrial Biotechnology
URN: urn:nbn:se:kth:diva-33427OAI: diva2:415406
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

Open Access in DiVA

No full text

Search in DiVA

By author/editor
Grimm, SebastianNygren, Per-Åke
By organisation
Molecular Biotechnology
Industrial Biotechnology

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Total: 52 hits
ReferencesLink to record
Permanent link

Direct link