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A small bispecific protein selected for orthogonal affinity purification
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0002-2643-8241
KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.ORCID iD: 0000-0002-6104-6446
KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
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2010 (English)In: BIOTECHNOL J, ISSN 1860-6768, Vol. 5, no 6, 605-617 p.Article in journal (Refereed) Published
Abstract [en]

A novel protein domain with dual affinity has been created by randomization and selection. The small alkali-stabilized albumin-binding domain (ABD(star)), used as scaffold to construct the library, has affinity to human serum albumin (HSA) and is constituted of 46 amino acids of which 11 were randomized. To achieve a dual binder, the binding site of the inherent HSA affinity was untouched and the randomization was made on the opposite side of the molecule. Despite its small size and randomization of almost a quarter of its amino acids, a bifunctional molecule, ABDz1, with ability to bind to both HSA and the Z(2) domain/protein A was successfully selected using phage display. Moreover, the newly selected variant showed improved affinity for HSA compared to the parental molecule. This novel protein domain has been characterized regarding secondary structure and affinity to the two different ligands. The possibility for affinity purification on two different matrices has been investigated using the two ligands, the HSA matrix and the protein A-based, MabSelect SuRe matrix, and the new protein domain was purified to homogeneity. Furthermore, gene fusions between the new domain and three different target proteins with different characteristics were made. To take advantage of both affinities, a purification strategy referred to as orthogonal affinity purification using two different matrices was created. Successful purification of all three versions was efficiently carried out using this strategy.

Place, publisher, year, edition, pages
2010. Vol. 5, no 6, 605-617 p.
Keyword [en]
Albumin binding domain, Bispecific binder, Orthogonal purification, Phage display, Z domain
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-27283DOI: 10.1002/biot.201000041ISI: 000278969600009PubMedID: 20518064OAI: oai:DiVA.org:kth-27283DiVA: diva2:377560
Note

QC 20101214

Available from: 2010-12-14 Created: 2010-12-09 Last updated: 2012-11-22Bibliographically approved
In thesis
1. Interaction engineered three-helix bundle domains for protein recovery and detection
Open this publication in new window or tab >>Interaction engineered three-helix bundle domains for protein recovery and detection
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

HTML clipboard The great advances in DNA technology, e.g. sequencing and recombinant DNA techniques, have given us the genetic information and the tools needed to effectively produce recombinant proteins. Recombinant proteins are valuable means in biotechnological applications and are also emerging as alternatives in therapeutic applications. Traditionally, monoclonal antibodies have been the natural choice for biotechnological and therapeutic applications due to their ability to bind a huge range of different molecules and their natural good affinity. However, the large size of antibodies (150 kDa) limits tissue penetration and the recombinant expression is complicated. Therefore, alternative binders with smaller sizes have been derived from antibodies and alternative scaffolds.

In this thesis, two structurally similar domains, Zbasic and ABDz1, have been used as purification tags in different contexts. They are both three-helical bundles and derived from bacterial surface domains, but share no sequence homology. Furthermore, by redesign of the scaffold used for ABDz1, a molecule intended for drug targeting with extended in-vivo half-life has been engineered. In Papers I and II, the poly-cationic tag Zbasic is explored and evaluated. Paper I describes the successful investigation of Zbasic as a purification handle under denaturating conditions. Moreover, Zbasic is evaluated as an interaction domain in matrixassisted refolding. Two different proteins were successfully refolded using the same setup without individual optimization. In Paper II, Zbasic is further explored as a purification handle under non-native conditions in a multi-parallel setup. In total, 22 proteins with varying characteristics are successfully purified using a multi-parallel protein purification protocol and a robotic system. Without modifications, the system can purify up to 60 proteins without manual handling. Paper I and II clearly demonstrate that Zbasic can be used as an interaction domain in matrix-assisted refolding and that it offers a good alternative to the commonly used His6-tag under denaturating conditions. In paper III, the small bifunctional ABDz1 is selected from a phage display library. Endowed with two different binding interfaces, ABDz1 is capable of binding both the HSA-sepharose and the protein A-derived MabSelect SuRe-matrix. The bifunctionality of the domain is exploited in an orthogonal affinity setup. Three target proteins are successfully purified using the HSA-matrix and the MabSelect SuRe-matrix. Furthermore, the purity of the target proteins is effectively improved by combining the two chromatographic steps. Thus, paper III shows that the small ABDz1 can be used as an effective purification handle and dual affinity tag without target specific optimization. Paper IV describes the selection and affinity maturation of small bispecific drug-targeting molecules. First generation binders against tumor necrosis factor-α are selected using phage display. Thereafter on-cell surface display and flow cytometry is used to select second-generation binders. The binding to tumor necrosis factor-α is improved up to 30 times as compared to the best first generation binder, and a 6-fold improvement of the binding strength was possible with retained HSA affinity. Paper III and IV clearly demonstrate that dual interaction surfaces can successfully be grafted on a small proteinaceous domain, and that the strategy in paper IV can be used for dual selection of bifunctional binders.

Place, publisher, year, edition, pages
Stockholm: KTH, 2010. x, 79 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2010:5
Keyword
ABD, Zbasic, albumin, protein engineering, phage display, staphylococcal display, inclusion bodies, refolding, proteomics, orthogonal affinity purification
National Category
Industrial Biotechnology Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-12823 (URN)978-91-7415-601-0 (ISBN)
Public defence
2010-05-28, D2, entréplan, Lindstedtsvägen 5, KTH, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC20100610

Available from: 2010-05-12 Created: 2010-05-12 Last updated: 2012-11-21Bibliographically approved
2. An albumin-binding domain as a scaffold for bispecific affinity proteins
Open this publication in new window or tab >>An albumin-binding domain as a scaffold for bispecific affinity proteins
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein engineering and in vitro selection systems are powerful methods to generate binding proteins. In nature, antibodies are the primary affinity proteins and their usefulness has led to a widespread use both in basic and applied research. By means of combinatorial protein engineering and protein library technology, smaller antibody fragments or alternative non-immunoglobulin protein scaffolds can be engineered for various functions based on molecular recognition. In this thesis, a 46 amino acid small albumin-binding domain derived from streptococcal protein G was evaluated as a scaffold for the generation of affinity proteins. Using protein engineering, the albumin binding has been complemented with a new binding interface localized to the opposite surface of this three-helical bundle domain. By using in vitro selection from a combinatorial library, bispecific protein domains with ability to recognize several different target proteins were generated. In paper I, a bispecific albumin-binding domain was selected by phage display and utilized as a purification tag for highly efficient affinity purification of fusion proteins. The results in paper II show how protein engineering, in vitro display and multi-parameter fluorescence-activated cell sorting can be used to accomplish the challenging task of incorporating two high affinity binding-sites, for albumin and tumor necrosis factor-alpha, into this new bispecific protein scaffold. Moreover, the native ability of this domain to bind serum albumin provides a useful characteristic that can be used to extend the plasma half-lives of proteins fused to it or potentially of the domain itself. When combined with a second targeting ability, a new molecular format with potential use in therapeutic applications is provided. The engineered binding proteins generated against the epidermal growth factor receptors 2 and 3 in papers III and IV are aimed in this direction. Over-expression of these receptors is associated with the development and progression of various cancers, and both are well-validated targets for therapy. Small bispecific binding proteins based on the albumin-binding domain could potentially contribute to this field. The new alternative protein scaffold described in this thesis is one of the smallest structured affinity proteins reported. The bispecific nature, with an inherent ability of the same domain to bind to serum albumin, is unique for this scaffold. These non-immunoglobulin binding proteins may provide several advantages as compared to antibodies in several applications, particularly when a small size and an extended half-life are of key importance. 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2012. x, 103 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2012:21
Keyword
albumin-binding domain, bispecific, albumin, affinity protein, phage display, staphylococcal display, orthogonal affinity purification, TNF-alpha, ErbB2, ErbB3
National Category
Biochemistry and Molecular Biology Medical Biotechnology
Research subject
SRA - Molecular Bioscience
Identifiers
urn:nbn:se:kth:diva-105425 (URN)978-91-7501-569-9 (ISBN)
Public defence
2012-12-14, FR4, AlbaNova University Center, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note

QC 20121122

Available from: 2012-11-22 Created: 2012-11-21 Last updated: 2012-11-22Bibliographically approved

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