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Strategies for functionalization of recombinant spider silk

Jansson, Ronnie (2015). Strategies for functionalization of recombinant spider silk. Diss. (sammanfattning/summary) Uppsala : Sveriges lantbruksuniv., Acta Universitatis agriculturae Sueciae, 1652-6880 ; 2015:11
ISBN 978-91-576-8220-8
eISBN 978-91-576-8221-5
[Doctoral thesis]

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Abstract

The use of silk-based materials for medical and biotechnological applications has been investigated for many years, with particular progress the last fifteen years. Extensive research has been conducted on silk derived from the silkworm Bombyx mori, but lately the evolvement in recombinant production has made mimics of spider silk proteins increasingly available. Revealed characteristics of silk such as biocompatibility, biodegradability and mechanical strength are features highly desirable in materials for medical purpose. This, in combination with techniques for functionalization (addition of new functions), incentivize further development of silk into highly sophisticated materials useful for advanced applications.

The main objective of this thesis has been to investigate novel strategies for functionalization of the recombinant spider silk protein 4RepCT. Two distinct approaches were used, coating and genetic fusion. We showed that coating of silk with polyelectrolytes could be employed to make the fibers electrically conductive as well as fluorescent. Parameters affecting the coating efficiency were investigated, and pH was shown to play an important role.

Genetic engineering was employed to fuse 4RepCT with moderately sized protein domains with inherent binding affinities for IgG, albumin and biotin, respectively. We found that the designed silk fusion proteins could self-assemble into silk-like fibers with preserved affinity of the added domains to bind their intended targets. Moreover, we could also demonstrate a general principle for presentation of biomolecules on IgG-binding spider silk by applying a two-step procedure, exemplified by presentation of VEGF via an anti-VEGF antibody bound to IgG-binding silk. We also showed a proof-of-principle for 4RepCT materials with catalytic activity by genetic linkage of an enzyme.

To investigate potential use of other heterologous hosts for increased production of functionalized silk fusion proteins, the methylotrophic yeast Pichia pastoris was evaluated. We demonstrated expression and secretion of an IgG-binding silk fusion protein in P. pastoris, although the protein was subjected to degradation as well as glycosylation.

Authors/Creators:Jansson, Ronnie
Title:Strategies for functionalization of recombinant spider silk
Series/Journal:Acta Universitatis agriculturae Sueciae (1652-6880)
Year of publishing :2015
Depositing date:2015
Volume:2015:11
Number of Pages:75
Papers/manuscripts:
NumberReferences
IMüller C., Jansson R., Elfwing A., Askarieh G., Karlsson R., Hamedi M., Rising A., Johansson J., Inganäs O. and Hedhammar M. (2011). Functionalisation of recombinant spider silk with conjugated polyelectrolytes. Journal of Materials Chemistry 21(9), 2909-2915.
IIJansson R., Thatikonda N., Lindberg D., Rising A., Johansson J., Nygren P.-Å. and Hedhammar M. (2014). Recombinant spider silk genetically functionalized with affinity domains. Biomacromolecules 15(5), 1696- 1706.
IIIJansson R., Hin Lau C., Ishida T., Ramström M., Sandgren M. and Hedhammar M. (2015). Expression of a functionalized spider silk protein in the methylotrophic yeast Pichia pastoris. Manuscript.
IVJansson R., Courtin C.M., Sandgren M. and Hedhammar M. (2015). Rational design of spider silk materials genetically functionalized with enzymatic activity. Manuscript.
Place of Publication:Uppsala
Publisher:Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences
ISBN for printed version:978-91-576-8220-8
ISBN for electronic version:978-91-576-8221-5
ISSN:1652-6880
Language:English
Publication Type:Doctoral thesis
Full Text Status:Public
Agris subject categories.:X Agricola extesions > X30 Life sciences
X Agricola extesions > X60 Technology
Subjects:(A) Swedish standard research categories 2011 > 1 Natural sciences > 106 Biological Sciences (Medical to be 3 and Agricultural to be 4) > Biochemistry and Molecular Biology
(A) Swedish standard research categories 2011 > 3 Medical and Health Sciences > 304 Medical Biotechnology > Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
(A) Swedish standard research categories 2011 > 3 Medical and Health Sciences > 304 Medical Biotechnology > Biomaterials Science
Agrovoc terms:silk, araneae, animal fibres, recombination, proteins, genes, pichia, yeasts, immunoglobulins, enzymes, immobilization
Keywords:spider silk, functionalization, gene fusion, affinity domain, IgG, biomolecule presentation, enzyme immobilization, conductivity, Pichia pastoris
URN:NBN:urn:nbn:se:slu:epsilon-e-2317
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-e-2317
ID Code:11753
Faculty:VH - Faculty of Veterinary Medicine and Animal Science
Department:(VH) > Dept. of Anatomy, Physiology and Biochemistry
Deposited By: Ronnie Jansson
Deposited On:22 Jan 2015 13:46
Metadata Last Modified:13 Apr 2015 14:00

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