Change search
ReferencesLink to record
Permanent link

Direct link
Ribosome display for selection and evolution of affibody molecules
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
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.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2011:9
Keyword [en]
affibody, ribosome display, phage display, combinatorial protein engineering, library, murine IgG1, SATB1, Ras, Raf
National Category
Other Industrial Biotechnology
Research subject
SRA - Molecular Bioscience
Identifiers
URN: urn:nbn:se:kth:diva-33191ISBN: 978-91-7415-952-3OAI: oai:DiVA.org:kth-33191DiVA: diva2:413896
Public defence
2011-05-27, Fr4, AlbaNova University Center, Roslagstullsbacken 21, B3, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20110506Available from: 2011-05-06 Created: 2011-04-29 Last updated: 2011-05-06Bibliographically approved
List of papers
1. Ribosome Display Selection of a Murine IgG(1) Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies
Open this publication in new window or tab >>Ribosome Display Selection of a Murine IgG(1) Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies
2011 (English)In: Molecular Biotechnology, ISSN 1073-6085, E-ISSN 1559-0305, Vol. 48, no 3, 263-276 p.Article in journal (Refereed) Published
Abstract [en]

Affinity reagents recognizing constant parts of antibody molecules are invaluable tools in immunotechnology applications, including purification, immobilization, and detection of immunoglobulins. In this article, murine IgG(1), the primary isotype of monoclonal antibodies (mAbs) was used as target for selection of novel binders from a combinatorial ribosome display (RD) library of 10(11) affibody molecules. Four rounds of selection using three different mouse IgG(1) mAbs as alternating targets resulted in the identification of binders with broad mIgG(1) recognition and dissociation constants (K (D)) in the low nanomolar to low micromolar range. For one of the binders, denoted Z(mab25), competition in binding to full length mIgG(1) by a streptococcal protein G (SPG) fragment and selective affinity capture of mouse IgG(1) Fab fragments after papain cleavage of a full mAb suggest that an epitope functionally overlapping with the SPG-binding site in the CH1 domain of mouse IgG(1) had been addressed. Interestingly, biosensor-based binding experiments showed that neither human IgG(1) nor bovine Ig, the latter present in fetal bovine serum (FBS) was recognized by Z(mab25). This selective binding profile towards murine IgG(1) was successfully exploited in species selective recovery of two different mouse mAbs from complex samples containing FBS, resembling a hybridoma culture supernatant.

Keyword
Mouse IgG1, Affibody, Selection, Fab fragment, Combinatorial protein engineering, Ribosome display
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-33435 (URN)10.1007/s12033-010-9367-1 (DOI)000291656700008 ()21197589 (PubMedID)2-s2.0-80054079725 (ScopusID)
Funder
Swedish Research Council, 50548301
Note
QC 20110705Available from: 2011-05-06 Created: 2011-05-06 Last updated: 2015-04-16Bibliographically approved
2. Single domain affinity proteins for the detection of the genome organizer protein SATB1
Open this publication in new window or tab >>Single domain affinity proteins for the detection of the genome organizer protein SATB1
Show others...
(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
affibody molecule, VHH antibody fragment, phage display, ribosome display, SATB1
Identifiers
urn:nbn:se:kth:diva-33425 (URN)
Funder
EU, FP7, Seventh Framework Programme, FLUODIAMON 201 837
Note
QS 2011Available from: 2011-05-06 Created: 2011-05-06 Last updated: 2011-05-06Bibliographically approved
3. Selection and characterisation of affibody molecules inhibiting the interaction between Ras and Raf in vitro
Open this publication in new window or tab >>Selection and characterisation of affibody molecules inhibiting the interaction between Ras and Raf in vitro
Show others...
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

National Category
Other Industrial Biotechnology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-30521 (URN)000285896200008 ()2-s2.0-78649713667 (ScopusID)
Funder
Swedish Research Council
Note
QC 20110304Available from: 2011-03-04 Created: 2011-02-28 Last updated: 2011-05-06Bibliographically approved
4. Affinity maturation of an affibody molecule binding to human Raf-1 via non-targeted in vitro evolution
Open this publication in new window or tab >>Affinity maturation of an affibody molecule binding to human Raf-1 via non-targeted in vitro evolution
(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
affibody molecule, ribosome display, error-prone PCR, library, evolution
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-33427 (URN)
Note
QS 2011Available from: 2011-05-06 Created: 2011-05-06 Last updated: 2011-05-06Bibliographically approved

Open Access in DiVA

Ribosome display for selection and evolution of affibody molecules(13483 kB)1660 downloads
File information
File name FULLTEXT02.pdfFile size 13483 kBChecksum SHA-512
4494888ecc4d97b1e62c0c7c05d2247d61339d4f382908636e905d63151c94f166acae23eee9549b7b8e900f87f05b6094afe9778e209fc6a87ad7d90a3aa1fb
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Grimm, Sebastian
By organisation
Molecular Biotechnology
Other Industrial Biotechnology

Search outside of DiVA

GoogleGoogle Scholar
Total: 1660 downloads
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: 271 hits
ReferencesLink to record
Permanent link

Direct link