Download Mr. David Cortens In Vivo Synthesis of ?Click? Functionalized

In Vivo Synthesis of “Click” Functionalized Nanobodies for
Advanced Biosensing Platforms
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D. Cortens , E. Steen Redeker , P. Adriaensens and W. Guedens
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Biomolecule Design Group, Institute for Materials Research (IMO), Hasselt University, BE3590 Diepenbeek, Belgium
Abstract
Immobilization of proteins on solid surfaces is of great importance in a large range of applications, e.g.
proteomics, drug screening and medical diagnostics. Most available strategies allow either the
formation of an oriented (spatially controlled) or a covalent coupling, but not both simultaneously (1).
This work is innovating due to the development and combination of different disciplines to obtain
spatially controlled and covalent coupling of proteins on solid surfaces for the production of advanced
biosensors. For the covalent coupling “click” chemistry is used. The introduction of “click” chemistry
into proteins is done by using a ‘nonsense suppression’. For this the genetic code of S. cerevisiae is
expanded with a genetically encoded, mutant, orthogonal E.coli aminoacyl-tRNA synthetase
(EcaaRS)/tRNACUA pair responsible for the incorporation of a “click” functionalized amino acid. The
benefit of this strategy is that it allows the production of proteins that contain a genetically encoded
orthogonal functional group (i.e. alkyne or azide) on a single, strategically chosen position in the
protein.
Keywords: site-specific modification, nonsense suppression, click chemistry, Nanobodies
1. INTRODUCTION
The site-specific modification of proteins plays an important role in current technologies. With the fast
progress and miniaturization of protein-based applications, a controlled orientation of proteins on solid
surfaces is required. Here we focus on the production of innovative, sensitive and cost-effective
biosensors. Using nonsense suppression in S. cerevisiae, an in vivo system is developed that enables
the site-specific incorporation of bioorthogonal functional groups ( e.g. “click functionalities) into
proteins (2). These “click” groups can perform different roles depending on the application. For
example, they can act as a unique chemical ‘handle’ for oriented and covalent immobilization of
proteins on complementary functionalized surfaces for the production of bioactive materials. Besides
this they can be used to introduce physical and fluorescent probes or NMR tags. In this research,
nanobodies will be used as a protein system, although this in vivo system is applicable for other types
of proteins as well. For the development of stable bio-active surfaces, nanobodies are very suited
proteins. The classical antibodies of mammals consist of two identical H-chains (heavy chains) and
two L-chains (light chains). The IgG antibodies of species of camilidae form an exception. Their serum
also contains a certain amount of so-called “heavy chain” antibodies (HCABs) (3,4)
In HCAbs the
antigen binding domain is reduced to a single variable domain, the VHH. The cloned and isolated VHH
domain, or Nanobody(Nb), is a very stable polypeptide and is the smallest intact antigen binding
fragment known (5). They are coded by a single gene, which makes them easy to manipulate. The fact
that they are very stable in a wide variety of conditions and heat (6), makes them very suitable for the
production of bio-active materials with a long shelf life.
2. METHODOLOGY
2.1 Nonsense Suppression
The in vivo system proposed here, is based on nonsense suppression. In nature, three stop codons
exist, UAG (Amber), UAA (Ochre) and UGA (Opal), causing the end of protein translation. Mutations
may occur as well, changing a codon into one of these stop codons, resulting in an early termination of
the polypeptide and a non- functional protein. Nonsense suppression is a mechanism found in nature
that suppresses this early termination. A nonsense suppressor is a tRNA gene containing a mutation
in its anticodon resulting in the recognition of one of the stop codons. In this project we choose the E.
Tyr
Coli tyrosine tRNACUA (tRNACUA ) containing a mutation in its anticodon (GUA → CUA). This tRNA
recognizes the amber codon and incorporates a tyrosine in response. Each tRNA is aminoacylated by
Tyr
its corresponding aminoacyl-tRNA-synthetase. In case of the tRNACUA
it is the tyrosyl-tRNAsynthetase (TyrRS). Within this project, TyrRS is modified to present high affinity for a non-natural,
“click” functionalized amino acid, more specific p-azidophenylalanine, instead of tyrosine. In
Tyr
combination with tRNACUA , it will incorporate this “click” modified amino acid as a response to an
amber stop codon. By introducing the amber codon at a well-chosen position, we can control the
Tyr
location of the modification. Since it is known that the E.coli TyrRS/tRNACUA pair does not interfere
with the natural transcriptional/ translational machinery of S. cerevisiae (2,7), it can be added to the
genetic repertoire of the yeast and therefore encode for a 21st (non-natural) amino acid.
2.2 The Use of S. Cerevisiae
Yeast combines the ease of microbial growth and the simplicity of manipulation with an eukaryotic
environment and the possibility to perform eukaryote specific posttranslational modifications (8). Even
though nanobodies do not require any posttranslational modification, we still choose to use yeast. The
idea behind this is to create a generic system that can also be used to site-specifically modify other
types of more complex proteins. An additional benefit is the fact that the translational machinery in
eukaryotic cells is strongly conserved. Therefore genes, involved in the site-specific incorporation of
non-natural amino acids in yeast, can most likely be used in higher organisms as well (9–11).
2.3 Click Chemistry
For the covalent attachment of the nanobodies “click” chemistry is used. In this project we make use of
the copper catalysed Huisgen 1,3-dipolar cycloaddition of azides and alkynes, although other “click”
reactions are possible as well (12,13). The trademark of a “click” reaction is the fact that it can easily
be performed in mild, physiological conditions, without the presence of unwanted site reactions. This
feature of “click” chemistry makes it very suitable to use for the immobilization of proteins, which
generally are very sensitive for their environment.
3. RESULTS
Incorporation of the unnatural amino acid was tested by
cytoplasmic expression of GFP, containing an amber codon at
location 48, in combination with the modified E.coli
Tyr
TyrRS/tRNACUA pair. The cells were grown in both the presence
and absence of the modified amino acid p-azidophenylalanine. In
case of successful amber suppression by the modified E.coli
Tyr
TyrRS/tRNACUA
pair, full length GFP will be expressed and
fluorescence will be visible. However, when amber suppression is
not occurring, it will result in an early termination of the polypeptide
and in a non- fluorescent GFP protein. Our results (Fig. 1) show
that only when p-azidophenylalanine is added to the growth Figure 1: pellet of MaV203 cells
containing pTEF/GFP_TAG and the
medium, full length GFP is expressed, indicating that the mutant modified E.coli TyrRS/tRNACUATyr pair.
EcTyrRS selectively incorporates p-azidophenylalanine in Left: without p-azidophenylalanine;
response to the amber codon. Nevertheless, additional right: with 1mM p-azidophenylalanine
added to the growth medium.
experiments need to be performed.
4. CONCLUSION
The main focus of this project is the development of innovative, sensitive and cheap biosensors which
are useful in numerous fields, e.g. health sector, environmental sector, food sector,…. An important
step in this process is the oriented coupling of receptor molecules on the surface. To overcome the
orientation and stability issues, an in vivo system is being developed to site-specifically incorporate
bioorthogonal functional groups into proteins that can act as a unique chemical ‘handle’ for oriented
and covalent immobilization. Our results suggest that nonsense suppression can be used to
incorporate non-natural amino acids on well-chosen locations, although more research is still needed
for valorisation.
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Acknowledgements
We want to thank the IWT for the funding of this project. We also want to thank the Interreg IV-A
project “BioMiMedics” (www.biomimedics.org) for the financial contribution from the EU and the
province of Limburg-Belgium.