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Recombinant Spider Silk Genetically Functionalized with Affinity Domains
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2014 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 15, no 5, p. 1696-1706Article in journal (Refereed) Published
Abstract [en]

Functionalization of biocompatible materials for presentation of active protein domains is an area of growing interest. Herein, we describe a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains of broad biotechnological use. Four affinity domains of different origin and structure; the IgG-binding domains Z and C2, the albumin-binding domain ABD, and the biotin-binding domain M4, were all successfully produced as soluble silk fusion proteins under nondenaturing purification conditions. Silk films and fibers produced from the fusion proteins were demonstrated to be chemically and thermally stable. Still, the bioactive domains are concluded to be folded and accessible, since their respective targets could be selectively captured from complex samples, including rabbit serum and human plasma. Interestingly, materials produced from mixtures of two different silk fusion proteins displayed combined binding properties, suggesting that tailor-made materials with desired stoichiometry and surface distributions of several binding domains can be produced. Further, use of the IgG binding ability as a general mean for presentation of desired biomolecules could be demonstrated for a human vascular endothelial growth factor (hVEGF) model system, via a first capture of anti-VEGF IgG to silk containing the Z-domain, followed by incubation with hVEGF. Taken together, this study demonstrates the potential of recombinant silk, genetically functionalized with affinity domains, for construction of biomaterials capable of presentation of almost any desired biomolecule.

Place, publisher, year, edition, pages
2014. Vol. 15, no 5, p. 1696-1706
Keywords [en]
Streptococcal Protein-G, Binding-Proteins, Fusion Proteins, Serum-Albumin, Fibroin, Cell, Fibers, Biomaterials, Antibody, Bundle
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-146554DOI: 10.1021/bm500114eISI: 000335939800016Scopus ID: 2-s2.0-84900422237OAI: oai:DiVA.org:kth-146554DiVA, id: diva2:724246
Funder
Swedish Research CouncilVinnova
Note

QC 20140612

Available from: 2014-06-12 Created: 2014-06-12 Last updated: 2018-10-02Bibliographically approved
In thesis
1. Functionalization of spider silk with affinity and bioactive domains via genetic engineering for in vitro disease diagnosis and tissue engineering
Open this publication in new window or tab >>Functionalization of spider silk with affinity and bioactive domains via genetic engineering for in vitro disease diagnosis and tissue engineering
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the recent past, spider silk has drawn significant attention from researchers mainly due to its distinguished mechanical strength, elasticity, biocompatibility and biodegradability. Technological advancements in genetic engineering have resulted in methods for creation of partial spider silk proteins. The main objective of this thesis has been to functionalize a partial spider silk protein, 4RepCT, with different affinity and bioactive domains via genetic engineering. Furthermore, the applicability of materials based on functionalized/bioactivated partial spider silk proteins for in vitro disease diagnosis and tissue engineering applications has been investigated.

In Paper I, four affinity domains of different sizes and folds were genetically attached to 4RepCT. All four silk fusion proteins could self-assemble to silk-like fibers. The retained ability of each added affinity domain to bind its respective target while in silk format was also verified. A construct where a monomeric streptavidin domain was genetically fused to 4RepCT was used to allow non-covalent presentation of biotinylated growth factors. Such materials have potential for applications where capture of growth factors could be advantageous, for example in vitro cell culture studies.

In Paper II, as a proof-of-concept, two recombinant antibody fragments (scFvs), previously shown to contribute to the candidate protein signature for diagnosing Systemic Lupus Erythematosus (SLE), were covalently attached to either ends of two types of partial spider silk proteins, 4RepCT and NTCT. All of the generated silk fusion proteins were shown able to self-assemble into fibres as well as defined spots in an array. Significantly higher target detection signal was reported from scFv-silk fusion proteins when compared to the same added amount of scFvs alone in micro- and nanoarrays. Thus, scFv-silk fusion proteins can be used as capture probes in the generation of sensitive diagnostic immunoassays for effective disease diagnosis.

In Paper III, bioactivation of 4RepCT with a pleiotropic growth factor, basic fibroblast growth factor (bFGF) was investigated. The generated silk-bFGF fusion protein retained the propensity to self-assemble into surface coatings and silk-like fibers. Maintained functionality of the silk-bFGF coating to bind FGFR receptor was confirmed using surface plasmon resonance studies. Moreover, with the aim to create an artificial ECM, silk-bFGF protein was combined with FN-silk, an engineered spider silk protein previously reported to support cell adhesion. Retained bioactivity of the bFGF was confirmed by culture of primary human endothelial cells on combined silk coatings and within combined silk fibers, even when cultured in medium containing low serum and no supplemented soluble growth factors. These findings highlight the use of combined silk coatings for in vitro cell culture, and combined silk fibers as a potential scaffold for tissue engineering applications.

In Paper IV, the possibility to genetically fuse two affibody-based VEGFR2 binders, Zdimer and Ztetramer, to 4RepCT was investigated. Maintained activity of added engineered affibodies was confirmed by receptor phosphorylation and cell proliferation studies. Furthermore, the possibility to create vessel-like structures within a FN-silk based cell scaffold containing Ztetramer-silk fibrils was reported. These findings highlight the future potential of herein developed silk based cell scaffolds in regenerative medicine.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2018. p. 70
Series
TRITA-CBH-FOU ; 2018:48
Keywords
affinity domains, artificial ECM, basic fibroblast growth factor, functionalization, mammalian cell culture, micro-and nano arrays, partial spider silk, single chain variable fragments
National Category
Medical and Health Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-235698 (URN)978-91-7729-970-7 (ISBN)
Public defence
2018-10-31, Oskar Kleins Auditorium, Roslagstullsbacken 21, AlbaNova University Center, Stockholm, 10:15 (English)
Opponent
Supervisors
Note

QC 20181003

Available from: 2018-10-03 Created: 2018-10-02 Last updated: 2018-10-03Bibliographically approved

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Nygren, Per-Åke

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