Design and analysis of peptide based nanoparticles

Raman, Senthilkumar. Design and analysis of peptide based nanoparticles. 2008, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: http://edoc.unibas.ch/diss/DissB_8404

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Viruses are naturally formed bionanoparticles (BNPs), ranging in size from 22-150 nm. Remarkably, small viruses are composed of one single protein chain folding into a capsid structure with icosahedral symmetry. The icosahedron is built up from 60 asymmetric units and is the largest closed shell in which every subunit is in an identical environment. It is characterized by 2-fold, 3-fold and 5-fold rotational symmetry axes. By superposition of different protein oligomerization domains onto the symmetry axes of the icosahedron, a nanoparticle with icosahedral symmetry can be designed. To test our concept, we have designed a peptide comprising a slightly modified form of a pentameric coiled-coil domain of cartilage oligomeric matrix protein (COMP) linked to a de novo designed trimeric coiled-coil domain as a single chain. These two different oligomeric domains were linked by two glycine residues to provide flexibility between them and were fixed in their relative orientation with an intramolecular disulfide bridge. Computer modeling predicted that such a design would result in an icosahedral peptide based nanoparticle with a diameter of 17 nm. We chemically synthesized the above designed peptide and performed refolding studies and biophysical characterization using analytical ultra centrifugation (AUC) and electron microscopy (EM), thereby showing the formation of icosahedral nanoparticles with a diameter of ~ 17 nm. Subsequently, we switched to recombinant expression of the designed peptide. Again, we performed refolding studies with the expressed peptide and biophysical characterization (AUC and EM), thereby showing the formation of icosahedral nanoparticles with a diameter of ~ 17 nm. In addition, we showed that during refolding, parameters such as ionic strength, pH of the refolding buffer and presence of glycerol influence the formation of icosahedral nanoparticles. Moreover, we observed icosahedral nanoparticles in conditions in which the formation of intramolecular disulfide bridges is not possible. This showed that even in the absence of intramolecular disulfide bridge the helices of the two different oligomeric domains like to be arranged close to each other, as this will favor the formation of icosahedral nanoparticles.
In parallel, we also modified the designed peptide to include charged residues at the
interface between the two oligomeric domains. The idea was to fix the relative orientation
between the two oligomeric domains through ionic interactions. The subsequent
expression, refolding studies and biophysical characterization showed the formation of
nanoparticles, but they were lacking icosahedral symmetry. This result showed that the
design has to be optimized further to obtain icosahedral nanoparticles.
Moreover, we wanted to study whether in our design principles oligomerization motifs
other than coiled-coils can be used to form icosahedral nanoparticles. Accordingly, we
used globular foldon domain as the trimerization domain and the COMP as the
pentamerization domain. The results showed the formation of nanoparticles, but they
were lacking icosahedral symmetry. In addition, in the above design we studied the effect
of linker region by increasing the liker region to four and six residues, but it did not help
in the formation of icosahedral nanoparticles. However, when the foldon domain
extended with the trimeric coiled-coil domain as a single trimerization domain and with
COMP as the penatmerization domain, we observed icosahedral nanoparticles.
Viruses are known for their induction of strong antibody (B-cell) mediated immune
response in the host even against the self-antigens. This property is conferred by the
repetitive arrangement of the antigens on their surface. Peptide based nanoparticles have
similar properties to viruses, such as self-assembly and most importantly the repetitive
arrangement of subunits (peptide based nanoparticles are composed of 60 identical
monomeric chains). Therefore, we wanted to use our system as a ‘repetitive antigen
display carrier’ in vaccination, as an alternative to viral and viral based platforms such as
virus like particles (VLPs). To this end, and in order to understand the effect of our
nanoparticle size on immune response, we built different constructs displaying the pilin
epitope of Pseudomonas pathogen at their C-terminus. Computer modeling indicated that
these constructs would form icosahedral nanoparticles of three sizes: small (18 nm),
medium (23 nm) and large (29 nm). From the refolding studies, we observed mostly
aggregation and precipitation of the nanoparticles.
This effect presumably due to interparticle cross-linking, by the cysteine residues of
pilin epitope which are present at the periphery of nanoparticles, as we observed
aggregation of small size icosahedral nanoparticles upon changing from reducing to
oxidizing condition. Immunization results, because of the aggregation and precipitation
behavior of nanoparticles, showed poor immune response. However, the immunization
results showed that our nanoparticles present the attached epitope in their native form, as
we observed binding against native pilin protein. Moreover, immunization results from
our laboratory using medium size nanoparticles displaying Salmonella epitope D2
showed promising results, as we got antibody titer values which were well comparable to
the values obtained with VLPs. This places our system alongside with VLPs, which are in
clinical trials as a carrier in vaccination.
Advisors:Aebi, Ueli
Committee Members:Burkhard, Peter and Herrmann, Harald
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Structural Biology (Aebi)
UniBasel Contributors:Aebi, Ueli and Burkhard, Peter
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:8404
Thesis status:Complete
Number of Pages:181
Identification Number:
edoc DOI:
Last Modified:22 Jan 2018 15:50
Deposited On:13 Feb 2009 16:37

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