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Interaction engineered three-helix bundle domains for protein recovery and detection
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0002-2643-8241
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 [en]
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: urn:nbn:se:kth:diva-12823ISBN: 978-91-7415-601-0 (print)OAI: oai:DiVA.org:kth-12823DiVA: diva2:319049
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
List of papers
1. Single-step recovery and solid-phase refolding of inclusion body proteins using a polycationic purification tag
Open this publication in new window or tab >>Single-step recovery and solid-phase refolding of inclusion body proteins using a polycationic purification tag
2006 (English)In: Biotechnology Journal, ISSN 1860-6768, Vol. 1, 187-196 p.Article in journal (Refereed) Published
Abstract [en]

A strategy for purification of inclusion body-forming proteins is described, in which the positively charged domain Z(basic) is used as a fusion partner for capture of denatured proteins on a cation exchange column. It is shown that the purification tag is selective under denaturing conditions. Furthermore, the new strategy for purification of proteins from inclusion bodies is compared with the commonly used method for purification of His(6)-tagged inclusion body proteins. Finally, the simple and effective means of target protein capture provided by the Z(basic) tag is further successfully explored for solid-phase refolding. This procedure has the inherited advantage of combining purification and refolding in one step and offers the advantage of eluting the concentrated product in a suitable buffer.

Keyword
Zbasic, Ion exchange chromatography, Purification of inclusion body proteins, Solid-phase refolding
Identifiers
urn:nbn:se:kth:diva-12815 (URN)10.1002/biot.200500023 (DOI)16892247 (PubMedID)2-s2.0-33750601043 (Scopus ID)
Note
QC20100622Available from: 2010-05-12 Created: 2010-05-12 Last updated: 2010-10-20Bibliographically approved
2. High-throughput protein purification under denaturating conditions by the use of cation exchange chromatography
Open this publication in new window or tab >>High-throughput protein purification under denaturating conditions by the use of cation exchange chromatography
2007 (English)In: Biotechnology Journal, ISSN 1860-6768, Vol. 2, 709-716 p.Article in journal (Refereed) Published
Abstract [en]

A high-throughput protein purification strategy using the polycationic Z(basic) tag has been developed. In order for the strategy to be useful both for soluble and less soluble proteins, a denaturating agent, urea, was used in all purification steps. First, four target proteins were genetically fused to the purification tag, Z(basic). These protein constructs were purified by cation exchange chromatography and eluted using a salt gradient. From the data achieved, a purification strategy was planned including stepwise elution to enable parallel protein purification using a laboratory robot. A protocol that includes all steps, equilibration of the chromatography resin, load of sample, wash, and elution, all without any manual handling steps, was handled by the laboratory robot. The program allows automated purification giving milligram amounts of pure recombinant protein of up to 60 cell lysates. In this study 22 different protein constructs, with different characteristics regarding pI and solubility, were successfully purified by the laboratory robot. The data show that Z(basic) can be used as a general purification tag also under denaturating conditions. Moreover, the strategy enables purification of proteins with different pI and solubility using ion exchange chromatography (IEXC). The procedure is highly reproducible and allows for high protein yield and purity and is therefore a good complement to the commonly used His(6)-tag.

Identifiers
urn:nbn:se:kth:diva-12816 (URN)10.1002/biot.200700060 (DOI)17492715 (PubMedID)
Note
QC20100622Available from: 2010-05-12 Created: 2010-05-12 Last updated: 2010-11-02Bibliographically approved
3. A small bispecific protein selected for orthogonal affinity purification
Open this publication in new window or tab >>A small bispecific protein selected for orthogonal affinity purification
Show others...
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.

Keyword
Albumin binding domain, Bispecific binder, Orthogonal purification, Phage display, Z domain
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-27283 (URN)10.1002/biot.201000041 (DOI)000278969600009 ()20518064 (PubMedID)
Note

QC 20101214

Available from: 2010-12-14 Created: 2010-12-09 Last updated: 2012-11-22Bibliographically approved
4. Engineering bispecificityinto a single albumin-binding domain aimed for drug-targeting and extended in vivo half-life extension.
Open this publication in new window or tab >>Engineering bispecificityinto a single albumin-binding domain aimed for drug-targeting and extended in vivo half-life extension.
(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:kth:diva-12818 (URN)
Note

QC20100622

Available from: 2010-05-12 Created: 2010-05-12 Last updated: 2012-09-20Bibliographically approved

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