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|Title: ||Designing clickable proteins|
|Authors: ||Steen Redeker, Erik|
|Issue Date: ||2012|
|Citation: ||Belgian-German (Macro)Molecular Meeting, Advanced Materials by Modular Strategies: From Synthesis to Industrial Applications, Houffalize, Belgium, 3-4 December 2012|
|Abstract: ||Many diagnostic and drug discovery applications require biomolecules to be immobilized on different types of
solid supports. In contrast to the enormous progress made in the immobilization of DNA, protein immobilization
is still a challenging task. Usually, proteins are immobilized on substrates via weak non-specific adsorption or
via the covalent reaction of naturally occurring chemical functionalities within the proteins with complementary
reactive groups on the solid supports. In both cases, the proteins are coupled to the surface in a random
orientation. Possibly, this results in a reduction of the protein’s biological activity. Site-specific and covalent
immobilization on the other hand, will result in coupled proteins that are oriented in a definite and controlled
fashion, resulting in an optimal bioactive surface. Different site-specific immobilization strategies have therefore
been developed in the last decade. These strategies require unique and mutually reactive groups on both the
protein and the solid surface.
The present study deals with the site-specific introduction of bioorthogonal groups on the protein side. This
bioorthogonal chemistry introduced should not interfere with the endogenous functional groups present in amino
acids and the coupling reactions should be possible to perform under mild conditions. An interesting solution can
be found in “click” chemistry. A well-known type of “click” chemistry is the Huisgen 1,3-dipolar cycloaddition
between alkynes and azides. These “click” functionalities, once introduced in a protein, can act as a unique
chemical ‘handle’ toward an oriented and covalent immobilization on the surface of interest.
Different methods for the site-specific introduction of chemical functionalities in proteins are used in our
Biomolecule Design Group (BDG), including Intein mediated Protein Ligation (IPL) and nonsense suppression.
IPL produces recombinant proteins with a bioorthogonal group at one of its termini. Here we present the
development of an IPL method that produces recombinant proteins with a bioorthogonal group at its C-terminus.
With nonsense suppression it is possible to introduce bioorthogonal amino acids in proteins at genetically
The results will be presented on the basis of the BDG’s work horse proteins, Maltose Binding Protein (MBP) and
the nanobody (Nb) BCII10, a Nb against β-lactamase. Nbs, being single-domain antibody fragments derived
from camelid antibodies, are very stable and relatively small. They are encoded by a single gene and have an
activity comparable to classical antibodies. This makes Nbs excellent tools for the development of bioactive
surfaces needed in for example biosensing devices.|
|Type: ||Conference Material|
|Appears in Collections: ||Research publications|
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