Evaluation of a generic in vivo approach for the site-specific modification of proteins with a 'click chemistry functionalized amino acid' for the development of innovative bioactive materials

Abstract

The site-specific modification of proteins is of great importance for the development and improvement of many biotechnological applications (e.g. biosensors). The focus of this work is the in vivo site-specific modification of proteins by means of amber suppression. As a result, a non-natural amino acid can be introduced to the protein, which can serve as a unique chemical “handle” to immobilize proteins in an oriented and covalent way, resulting in a bioactive surface. The model protein used in this work is the nanobody BCII10. Nanobodies are very stable single-domain antibody fragments derived from camelid heavy-chain antibodies (HCAbs). They are relatively small proteins encoded by a single gene with an activity comparable to classical antibodies. In this work the yeast Saccharomyces cerevisiae (S. cerevisiae) is used for the production of the modified nanobody. Yeast combines the ease of microbial growth with an eukaryotic environment, enabling the possibility to introduce eukaryotic specific modifications, such as protein folding, disulfide bond formation, proteolytic processing and glycosylation. Besides, the translational machinery of eukaryotes is very well conserved, allowing genes involved in the site-specific incorporation of non-natural amino acids in yeast, to be duplicated and used in higher eukaryotes (e.g. mammalian cells) or other yeast species such as Pichia pastoris, Kluyveromyces lactis, Schizoschaccharomyces pombe and more. Amber suppression allows the incorporation of bioorthogonal functionalities (e.g. azide) in the form of a modified (non-natural) amino acid in response to an amber codon. For this purpose, the translational machinery of S. cerevisiae is expanded with a genetically encoded E. coli suppressor tRNACUATyr and a mutant E. coli tyrosyl-tRNA synthethase (EcTyrRS) with an affinity for p-azidophenylalanine (p-azidoPhe) instead of tyrosine. This will result in the incorporation of p-azidoPhe in response of the amber codon. Such a mutant EcTyrRS is obtained by the construction and screening of a library of mutant EcTyrRSs. Both the benefits as well as the difficulties of constructing such a library is discussed in chapter 5. The main advantage of the amber suppression approach is that it allows the production of proteins that contain a genetically encoded orthogonal functional group (i.e. azide) on a single, strategically chosen position in the protein. This then allows the oriented coupling of the ‘click’ functionalized nanobody to an alkynylated surface by means of the Huisgen 1,3-dipolar cycloaddition. Nevertheless, during the development of this work, two inconsistencies were encountered: one for the library screening and one for the protein expression. The problems will be briefly explained as follows. First, in order to obtain a mutant EcTyrRS, a library needs to be generated by a random mutation of certain amino acids present in the binding pocket of EcTyrRS. This library is then screened by means of a double-sieve method in the haploid yeast strain MaV203 to obtain the mutant EcTyrRS selective for the modified amino acid. The double-sieve method is based on the activation and repression of the URA3 gene in MaV203. The SPAL10::URA3 fusion in MaV203 cells was developed by Vidal et al. to actively repress URA3 gene expression unless full-length GAL4 is present. However, this work showed that spontaneous, recessive mutations occur in a single repressor gene of the SPAL10 promoter in MaV203 cells, resulting in a URA3+ phenotype without the presence of GAL4. This unwanted URA3 expression has a negative impact on the library screening. In this work evidence is provided that diploid cells can be used for the library screening to circumvent the unwanted activation of the URA3 gene (chapter 4). A second inconsistency was encountered during the protein expression experiments. Literature reports that yeast episomal plasmids (YEp) can be used to express both the EcTyrRS/tRNACUATyr pair as well as the protein of interest, containing the amber codon. However, the results discussed in chapter 6 of this work suggest that yeast episomal plasmids are not suited for the production of modified proteins and that more stable expression systems (e.g. yeast integrating plasmids) need to be investigated.