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In Vivo Synthesis of “Click” Functionalized Nanobodies for Advanced Biosensing Platforms 1 1 1 D. Cortens , E. Steen Redeker , P. Adriaensens and W. Guedens 1 1 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. 5. REFERENCES 1. Steen Redeker E, Ta DT, Cortens D, Billen B, Guedens W, Adriaensens P. Protein engineering for directed immobilization. Bioconjug Chem. 2013;24:1761–77. 2. Chin JW, Cropp TA, Chu S, Meggers E, Schultz PG, Jolla L. Progress Toward an Expanded Eukaryotic Genetic Code. Science (80- ). 2003;10:511–9. 3. Muyldermans S, Baral TN, Retamozzo VC, De Baetselier P, De Genst E, Kinne J, et al. Camelid immunoglobulins and nanobody technology. Vet Immunol Immunopathol [Internet]. 2009 Mar 15 [cited 2012 Mar 10];128(1-3):178–83. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19026455 4. Woolven BP, Frenken LGJ, van der Logt P, Nicholls PJ. The structure of the Ilama heavy chain constant genes reveals a mechanism for heavy-chain antibody formation. 1999;98–101. Available from: http://dx.doi.org/10.1007/s002510050694 5. Muyldermans S, Atarhouch T, Saldanha J, Barbosa J a, Hamers R. Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. Protein Eng. 1994;7(9):1129–35. 6. De Genst E, Saerens D, Muyldermans S, Conrath K. Antibody repertoire development in camelids. Dev Comp Immunol [Internet]. 2006 Jan [cited 2012 Mar 10];30(1-2):187–98. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16051357 7. Edwards H, Schimmel P. A bacterial amber suppressor in Saccharomyces cerevisiae is selectively recognized by a bacterial aminoacyl-tRNA synthetase. Mol Cell Biol [Internet]. 1990 Apr;10(4):1633–41. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=362268&tool=pmcentrez&rendertype =abstract 8. Eckart MR, Bussineau CM. Quality and authenticity of heterologous proteins synthesized in yeast. Curr Opin Biotechnol [Internet]. 1996 Oct;7(5):525–30. Available from: http://www.ncbi.nlm.nih.gov/pubmed/8939630 9. Köhrer C, Xie L, Kellerer S, Varshney U, RajBhandary UL. Import of amber and ochre suppressor tRNAs into mammalian cells: a general approach to site-specific insertion of amino acid analogues into proteins. Proc Natl Acad Sci U S A [Internet]. 2001 Dec 4;98(25):14310–5. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=64678&tool=pmcentrez&rendertype =abstract 10. Sakamoto K, Hayashi A, Sakamoto A, Kiga D, Nakayama H, Soma A, et al. Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells. Nucleic Acids Res [Internet]. 2002 Nov 1;30(21):4692–9. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=135798&tool=pmcentrez&rendertype =abstract 11. Liu CC, Schultz PG. Adding new chemistries to the genetic code. Annu Rev Biochem [Internet]. 2010 Jan [cited 2012 Mar 3];79(March):413–44. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20307192 12. Prescher J a, Bertozzi CR. Chemistry in living systems. Nat Chem Biol [Internet]. 2005 Jun;1(1):13–21. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16407987 13. Sletten EM, Bertozzi CR. Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angew Chem Int Ed Engl [Internet]. 2009 Jan [cited 2012 Mar 13];48(38):6974– 98. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2864149&tool=pmcentrez&rendertyp e=abstract 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.