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Chemical Engineering of Small Affinity Proteins
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0002-9969-0317
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Small robust affinity proteins have shown great potential for use in therapy, in vivo diagnostics, and various biotechnological applications. However, the affinity proteins often need to be modified or functionalized to be successful in many of these applications. The use of chemical synthesis for the production of the proteins can allow for site-directed functionalization not achievable by recombinant routes, including incorporation of unnatural building blocks. This thesis focuses on chemical engineering of Affibody molecules and an albumin binding domain (ABD), which both are three-helix bundle proteins of 58 and 46 amino acids, respectively, possible to synthesize using solid phase peptide synthesis (SPPS).

In the first project, an alternative synthetic route for Affibody molecules using a fragment condensation approach was investigated. This was achieved by using native chemical ligation (NCL) for the condensation reaction, yielding a native peptide bond at the site of ligation. The constant third helix of Affibody molecules enables a combinatorial approach for the preparation of a panel of different Affibody molecules, demonstrated by the synthesis of three different Affibody molecules using the same helix 3 (paper I).

In the next two projects, an Affibody molecule targeting the amyloid-beta peptide, involved in Alzheimer’s disease, was engineered. Initially the N-terminus of the Affibody molecule was shortened resulting in a considerably higher synthetic yield and higher binding affinity to the target peptide (paper II). This improved variant of the Affibody molecule was then further engineered in the next project, where a fluorescently silent variant was developed and successfully used as a tool to lock the amyloid-beta peptide in a β-hairpin conformation during studies of copper binding using fluorescence spectroscopy (paper III).

In the last two projects, synthetic variants of ABD, interesting for use as in vivo half-life extending partners to therapeutic proteins, were engineered. In the first project the possibility to covalently link a bioactive peptide, GLP-1, to the domain was investigated. This was achieved by site-specific thioether bridge-mediated cross-linking of the molecules via a polyethylene glycol (PEG)-based spacer. The conjugate showed retained high binding affinity to human serum albumin (HSA) and a biological activity comparable to a reference GLP-1 peptide (paper IV). In the last project, the possibility to increase the proteolytic stability of ABD through intramolecular cross-linking, to facilitate its use in e.g. oral drug delivery applications, was investigated. A tethered variant of ABD showed increased thermal stability and a considerably higher proteolytic stability towards pepsin, trypsin and chymotrypsin, three important proteases found in the gastrointestinal (GI) tract (paper V).

Taken together, the work presented in this thesis illustrates the potential of using chemical synthesis approaches in protein engineering.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. , viii, 79 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:3
Keyword [en]
Affibody molecules, albumin binding domain, ligation, protein synthesis, solid phase peptide synthesis
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-141014ISBN: 978-91-7595-004-4 (print)OAI: oai:DiVA.org:kth-141014DiVA: diva2:693824
Public defence
2014-03-07, FR4 (Oskar Klein), AlbaNova Universitetscentrum, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20140207

Available from: 2014-02-07 Created: 2014-02-05 Last updated: 2014-02-07Bibliographically approved
List of papers
1. A Native Chemical Ligation Approach for Combinatorial Assembly of Affibody Molecules
Open this publication in new window or tab >>A Native Chemical Ligation Approach for Combinatorial Assembly of Affibody Molecules
2012 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 13, no 7, 1024-1031 p.Article in journal (Refereed) Published
Abstract [en]

Affinity molecules labeled with different reporter groups, such as fluorophores or radionuclides, are valuable research tools used in a variety of applications. One class of engineered affinity proteins is Affibody molecules, which are small (6.5 kDa) proteins that can be produced by solid phase peptide synthesis (SPPS), thereby allowing site-specific incorporation of reporter groups during synthesis. The Affibody molecules are triple-helix proteins composed of a variable part, which gives the protein its binding specificity, and a constant part, which is identical for all Affibody molecules. In the present study, native chemical ligation (NCL) has been applied for combinatorial assembly of Affibody molecules from peptide fragments produced by Fmoc SPPS. The concept is demonstrated for the synthesis of three different Affibody molecules. The cysteine residue introduced at the site of ligation can be used for directed immobilization and does not interfere with the function of the investigated proteins. This strategy combines a high-yield production method with facilitated preparation of proteins with different C-terminal modifications.

Keyword
antibody mimics, biotechnology, chemoselectivity, peptides, solid-phase synthesis
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-95231 (URN)10.1002/cbic.201200052 (DOI)000303191900014 ()2-s2.0-84860251745 (Scopus ID)
Note

QC 20120524

Available from: 2012-05-24 Created: 2012-05-21 Last updated: 2017-12-07Bibliographically approved
2. N-terminal engineering of amyloid-beta-binding Affibody molecules yields improved chemical synthesis and higher binding affinity
Open this publication in new window or tab >>N-terminal engineering of amyloid-beta-binding Affibody molecules yields improved chemical synthesis and higher binding affinity
Show others...
2010 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 19, no 12, 2319-2329 p.Article in journal (Refereed) Published
Abstract [en]

The aggregation of amyloid-beta (A beta) peptides is believed to be a major factor in the onset and progression of Alzheimer's disease Molecules binding with high affinity and selectivity to A beta-peptides are important tools for investigating the aggregation process An A beta-binding Affibody molecule, Z(A beta 3), has earlier been selected by phage display and shown to bind A beta(1-40) with nanomolar affinity and to inhibit A beta-peptide aggregation In this study, we create truncated functional versions of the Z(A beta 3) Affibody molecule better suited for chemical synthesis production Engineered Affibody molecules of different length were produced by solid phase peptide synthesis and allowed to form covalently linked homodimers by S-S-bridges The N-terminally truncated Affibody molecules Z(A beta 3)(12-58), Z(A beta 3)(15-58), and Z(A beta 3)(18-58) were produced in considerably higher synthetic yield than the corresponding full-length molecule Z(A beta 3)(1-58) Circular dichroism spectroscopy and surface plasmon resonance-based biosensor analysis showed that the shortest Affibody molecule, Z(A beta 3)(18-58), exhibited complete loss of binding to the A beta(1-40)-peptide, while the Z(A beta 3)(12-58) and Z(A beta 3)(15-58) Affibody molecules both displayed approximately one order of magnitude higher binding affinity to the A beta(1-40)-peptide compared to the full-length Affibody molecule Nuclear magnetic resonance spectroscopy showed that the structure of A beta(1-40) in complex with the truncated Affibody dimers is very similar to the previously published solution structure of the A beta(1-40)-peptide in complex with the full-length Z(A beta 3) Affibody molecule This indicates that the N-terminally truncated Affibody molecules Z(A beta 3)(12-58) and Z(A beta 3)(15-58) are highly promising for further engineering and future use as binding agents to monomeric A beta(1-40)

Keyword
amyloid, protein engineering, Alzheimer's disease, solid phase peptide synthesis, NMR spectroscopy
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-27658 (URN)10.1002/pro.511 (DOI)000284793800006 ()2-s2.0-78649668010 (Scopus ID)
Funder
Knut and Alice Wallenberg Foundation
Note

QC 20101223

Available from: 2010-12-23 Created: 2010-12-20 Last updated: 2017-12-11Bibliographically approved
3. Engineered non-fluorescent Affibody molecules facilitate studies of the amyloid-beta (A beta) peptide in monomeric form: Low pH was found to reduce A beta/Cu(II) binding affinity
Open this publication in new window or tab >>Engineered non-fluorescent Affibody molecules facilitate studies of the amyloid-beta (A beta) peptide in monomeric form: Low pH was found to reduce A beta/Cu(II) binding affinity
Show others...
2013 (English)In: Journal of Inorganic Biochemistry, ISSN 0162-0134, E-ISSN 1873-3344, Vol. 120, 18-23 p.Article in journal (Refereed) Published
Abstract [en]

Aggregation of amyloid-beta (A beta) peptides into oligomers and amyloid plaques in the human brain is considered a causative factor in Alzheimer's disease (AD). As metal ions are over-represented in AD patient brains, and as distinct A beta aggregation pathways in presence of Cu(II) have been demonstrated, metal binding to A beta likely affects AD progression. A beta aggregation is moreover pH-dependent, and AD appears to involve inflammatory conditions leading to physiological acidosis. Although metal binding specificity to A beta varies at different pH's, metal binding affinity to A beta has so far not been quantitatively investigated at sub-neutral pH levels. This may be explained by the difficulties involved in studying monomeric peptide properties under aggregation-promoting conditions. We have recently devised a modified Affibody molecule, Z(A beta 3)(12-58), that binds A beta with sub-nanomolar affinity, thereby locking the peptide in monomeric form without affecting the N-terminal region where metal ions bind. Here, we introduce non-fluorescent A beta-binding Affibody variants that keep A beta monomeric while only slightly affecting the A beta peptide's metal binding properties. Using fluorescence spectroscopy, we demonstrate that Cu(II)/A beta(1-40) binding is almost two orders of magnitude weaker at pH 5.0 (apparent K-D = 51 mu M) than at pH 7.3 (apparent K-D = 0.86 mu M). This effect is arguably caused by protonation of the histidines involved in the metal ligandation. Our results indicate that engineered variants of Affibody molecules are useful for studying metal-binding and other properties of monomeric A beta under various physiological conditions, which will improve our understanding of the molecular mechanisms involved in AD.

Keyword
Alzheimer's disease, Affibody molecule, Copper ion, Binding constant, Protein engineering, Peptide aggregation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-119725 (URN)10.1016/j.jinorgbio.2012.11.005 (DOI)000315252300003 ()2-s2.0-84871389016 (Scopus ID)
Funder
Swedish Research CouncilVinnova
Note

QC 20130325

Available from: 2013-03-25 Created: 2013-03-21 Last updated: 2017-12-06Bibliographically approved
4. A GLP-1 receptor agonist conjugated to an albumin-binding domain for extended half-life
Open this publication in new window or tab >>A GLP-1 receptor agonist conjugated to an albumin-binding domain for extended half-life
Show others...
2014 (English)In: Biopolymers, ISSN 0006-3525, E-ISSN 1097-0282, Vol. 102, no 3, 252-259 p.Article in journal (Refereed) Published
Abstract [en]

Glucagon-like peptide 1 (GLP-1) and related peptide agonists have been extensively investigated for glycaemic control in Type 2 diabetes, and may also have therapeutic applications for other diseases. Due to the short half-life (t1/2<2 min) of the endogenous peptide, caused by proteolytic degradation and renal clearance, different strategies for half-life extension and sustained release have been explored. In the present study, conjugates between a GLP-1 analogue and a 5 kDa albumin-binding domain (ABD) derived from streptococcal protein G have been chemically synthesized and evaluated. ABD binds with high affinity to human serum albumin, which is highly abundant in plasma and functions as a drug carrier in the circulation. Three different GLP-1-ABD conjugates, with the two peptides connected by linkers of two, four, and six PEG units, respectively, were synthesized and tested in mouse pancreatic islets at high (11 mM) and low (3 mM) glucose concentration. Insulin release upon stimulation was shown to be glucose-dependent, showing no significant difference between the three different GLP-1-ABD conjugates and unconjugated GLP-1 analogue. The biological activity, in combination with the high affinity binding to albumin, make the GLP-1-ABD conjugates promising GLP-1 receptor agonists expected to show extended in vivo half-life.

Keyword
glucagon-like peptide 1, albumin-binding domain, half-life extension, peptide conjugate, insulin release
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-141009 (URN)10.1002/bip.22474 (DOI)000337513400004 ()2-s2.0-84922005237 (Scopus ID)
Note

Updated from Manuscript to Journal. QC 20140711

Available from: 2014-02-05 Created: 2014-02-05 Last updated: 2017-12-06Bibliographically approved
5. Intramolecular thioether cross-linking of therapeutic proteins to increase proteolytic stability
Open this publication in new window or tab >>Intramolecular thioether cross-linking of therapeutic proteins to increase proteolytic stability
2014 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 15, no 14, 2132-2138 p.Article in journal (Other academic) Published
Abstract [en]

Protein-based pharmaceuticals typically display high selectivity and low toxicity, but are also characterized by low oral availability, mainly because of enzymatic degradation in the gastrointestinal tract and poor permeability across the intestinal wall. One way to increase the proteolytic stability of peptides and proteins is by intramolecular crosslinking, such as the introduction of disulfide bridges. However, disulfide bridges are at risk of thiol-disulfide exchange or reduction during production, purification, and/or therapeutic use, whereas thioether bridges are expected to be stable under the same conditions. In this study, thioether crosslinking was investigated for a 46 aa albumin-binding domain (ABD) derived from streptococcal protein G. ABD binds with high affinity to human serum albumin (HSA) and has been proposed as a fusion partner to increase the in vivo half-lives of therapeutic proteins. In the study, five ABD variants with single or double intramolecular thioether bridges were designed and synthesized. The binding affinity, secondary structure, and thermal stability of each protein was investigated by SPR-based biosensor analysis and CD spectroscopy. The proteolytic stability in the presence of the major intestinal proteases pepsin (found in the stomach) and trypsin in combination with chymotrypsin (found in pancreatin secreted to the duodenum by the pancreas) was also investigated. The most promising crosslinked variant, ABD_CL1, showed high thermal stability, retained high affinity in binding to HSA, and showed dramatically increased stability in the presence of pepsin and trypsin/chymotrypsin, compared to the ABD reference protein. This suggests that the intramolecular thioether crosslinking strategy can be used to increase the stability towards gastrointestinal enzymes.

Keyword
Albumin binding domain, solid phase peptide synthesis, proteolytic stability, oral delivery
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-141013 (URN)10.1002/cbic.201400002 (DOI)000342807100017 ()2-s2.0-84908153975 (Scopus ID)
Funder
Swedish Research Council
Note

Updated from "Manuscript" to "Article in Journal". QC 20141112

Available from: 2014-02-05 Created: 2014-02-05 Last updated: 2017-12-06Bibliographically approved

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