Change search
Link to record
Permanent link

Direct link
BETA
Publications (4 of 4) Show all publications
Nilsson, A., Lindgren, J. & Eriksson Karlström, A. (2017). Intramolecular Thioether Crosslinking to Increase the Proteolytic Stability of Affibody Molecules. ChemBioChem (Print), 18(20), 2056-2062
Open this publication in new window or tab >>Intramolecular Thioether Crosslinking to Increase the Proteolytic Stability of Affibody Molecules
2017 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 18, no 20, p. 2056-2062Article in journal (Refereed) Published
Abstract [en]

Protein therapeutics suffer from low oral bioavailability, mainly due to poor membrane permeability and digestion by gastrointestinal proteases. To improve proteolytic stability, intramolecular thioether crosslinks were introduced into a three-helix affibody molecule binding the human epidermal growth factor receptor (EGFR). Solid-phase peptide synthesis was used to produce an unmodified control protein domain and three different crosslinked protein domain variants: one with a thioether crosslink between the N-terminal lysine residue and a cysteine residue in the second loop region (denoted K4), a second with a crosslink between the C-terminal lysine residue and a cysteine residue in the first loop region (denoted K58), and a third with crosslinks in both positions (denoted K4K58). Circular dichroism (CD) and surface-plasmon-resonance-based (SPR-based) biosensor studies of the protein domains showed that the three-helix structure and high-affinity binding to EGFR were preserved in the crosslinked protein domains. In vitro digestion by gastrointestinal proteases demonstrated that the crosslinked protein domains showed increased stability towards pepsin and towards a combination of trypsin and chymotrypsin.

Place, publisher, year, edition, pages
WILEY-V C H VERLAG GMBH, 2017
Keywords
affibodies, crosslinking, protein engineering, protein modifications, thioethers
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-217433 (URN)10.1002/cbic.201700350 (DOI)000413353800015 ()28836374 (PubMedID)2-s2.0-85030102142 (Scopus ID)
Note

QC 20171117

Available from: 2017-11-17 Created: 2017-11-17 Last updated: 2017-11-17Bibliographically approved
Lindgren, J., Refai, E., Zaitsev, S., Abrahmsén, L., Berggren, P.-O. & Eriksson Karlström, A. (2014). A GLP-1 receptor agonist conjugated to an albumin-binding domain for extended half-life. Biopolymers, 102(3), 252-259
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, p. 252-259Article 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.

Keywords
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
Lindgren, J. (2014). Chemical Engineering of Small Affinity Proteins. (Doctoral dissertation). Stockholm: KTH Royal Institute of Technology
Open this publication in new window or tab >>Chemical Engineering of Small Affinity Proteins
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. p. viii, 79
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:3
Keywords
Affibody molecules, albumin binding domain, ligation, protein synthesis, solid phase peptide synthesis
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-141014 (URN)978-91-7595-004-4 (ISBN)
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
Lindgren, J. & Eriksson Karlström, A. (2014). Intramolecular thioether cross-linking of therapeutic proteins to increase proteolytic stability. ChemBioChem (Print), 15(14), 2132-2138
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, p. 2132-2138Article 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.

Keywords
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
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-9969-0317

Search in DiVA

Show all publications