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10 Important Paper


Budisa, N., Minks, C., Medrano, F. J., Lutz, J., Huber, R., Moroder, L. (1998). Residue-specific bio-incorporation of non-natural biologically active amino acids into proteins as possible drug carriers. Structure and stability of per-thiaproline mutant or annexin V.
Proc. Acad. Sci., USA 95, 455-459

Engineering protein-based drug delivery: Built-in drugs could target tissues  
"This is very nice work," says Peter G. Schultz of the University of California, Berkeley. "It's a novel application of the incorporation of amino acid analogs into proteins."  
see Link 
“Instead of being packaged inside a pill, a special class of drugs can be stitched right into the fabric of a protein, a new study suggests. By choosing an appropriate protein as a delivery vehicle, scientists may be able to send these drugs to specific tissues or time their release in a new way. Researchers (....) demonstrated the feasibility of this idea by synthesizing a protein in which one amino acid was replaced with a non-natural, biologically active one. According to their scenario, the modified protein would travel inside the body like its normal counterpart and then deliver its drug--the nonstandard amino acid--to target cells.”(From: "Built-in drugs could target tissues." The Free Library. 1998 Science Service, Inc.). We elaborated this idea in more detail in paper published two years later (Tetrahedron, 2000, 56, 9431-9442;In vivo building and folding of protein shuttles for drug delivery and targeting by the selective pressure incorporation”).


Renner, C., Alefelder, S., Bae, J. H., Budisa, N., Huber, R., Moroder, L. (2001). Fluoroprolines as tools for protein design and engineering. Angew. Chem. Int. Ed. Engl. 40, 923-925.

Discovery of the role of proline puckering in the protein folding and translation.
  Here, we have reported the first in vivo protein translation with the fluorinated prolines. This study confirmed our expectation that there should be the direct correlation of folding of the biosynthesized protein with the conformational properties of incorporated Pro-analogs (e.g. cis/trans ratio, pKa, pyramidalization of the acceptor carbonyl group etc.). In following years, we have extended our studies on other proteins as well. In the year 2008 we published discovery of ‘superfolded’ green florescent protein’ along with its high-resolution structure  
(link) that lead us to discovery of the role of proline puckering in the global folding of the proteins. Our discovery revealed Pro-puckering as a new dimension/parameter in the protein folding nowadays conceptualized as (n -π*) interactions as an underlying principle behind the puckering. Most recently, we extended these studies to in vivo translation of synthetic Pro analogs with Pro-rich sequences and have discovered that there is strong bias during in vivo translation depending on stereo-chemical nature of the analog used, This lead us into current research about the fundamental chemical mechanisms behind the protein biosynthesis with oligo-prolines.

Bae, J., Rubini, M., Jung, G., Wiegand, G., Seifert, M. H. J., Azim, M. K., Kim, J. S., Zumbusch, A., Holak, T. A., Moroder, L., Huber, R., Budisa, N. (2003). Expansion of the Genetic Code Enables Design of a Novel "Gold" Class of Green Fluorescent Proteins.
J. Mol. Biol. 328, 977-1202.

Siehe auch: Lepthien, S., Hoesl, M. G., Merkel, L., Budisa, N. (2008). Azatryptophans endow proteins with intrinsic blue fluorescence.
Proc. Natl. Acad. Sci. USA.105, 16095-16100.

Making ‘gold’ from ‘turquoise’: Engineering of a new ‘golden’ class of synthetic auto-fluorescent proteins; efforts to design intrinsically colored proteins  
Here, we have reported a spectacular engineering of "gold-fluorescent protein" which represents a novel class of synthetic auto-florescent proteins generated by using non-canonical amino acids incorporation. It is most red-shifted GFP-protein (with emission maximum at 576 nm) among all Aequorea victoria-derived variants and mutants. In addition, we introduced GFP as the model protein in the field; nowadays, it is most commonly used model protein for monitoring and analysis of incorporation of non-canonical amino acids).

Wolschner, C., Giese, A., Kretzschmar, H., Huber, R., Moroder, L., Budisa, N. (2009). Design of anti- and pro-aggregation variants to assess the effects of methionine oxidation in human prion protein.
Proc. Natl. Acad. Sci. USA 106, 7756-7761.

Here we reported for the first time a chemical model that revealed that the methionine oxidation could be one of the major reasons for the conformational change in the prion protein during oxidative stress. This insight was gained through the design of aggregation-resistant and aggregation-prone prion protein variants by using noncanonical amino acids norleucine and metoxinine as methionine analogs. In particular, in collaboration with Kretschmar Group from LMU Munich, we examined the role of the chemical nature of Met-side chain in the pathogenesis of prion diseases. Surprisingly, it turned out that the methionine oxidation and oxidative stress is closely linked. We developed a chemical model that has led to the design of an aggregation-resistant prion protein. Through the separate incorporation of Nle and Mox we were able to gain solid data that the methionine oxidation could be one of the main reasons for the conformational change in the prion protein (in the context of oxidative stress). (more details see News from 2009 on our web site).


Hoesl, M. G., Acevedo Rocha, C., Nehring, S., Wolschner, C., Royter, M., Wiltschi, B., Budisa, N., Antranikian, G. (2011). Lipase Congeners Designed by Genetic Code Engineering. ChemCatChem, 3, 213-221.
Here we demonstrated, for the first time, the practical importance of the incorporation of noncanonical amino acids (ncAAs) for the field of biocatalysis. With the advent of recombinant DNA technology, the optimization of enzymes via rational and directed evolutionary approaches became the dominant route for the generation of sequence diversity. However, the task of identifying beneficial diversity is non-trivial, and mutations that span the entire structure of the enzyme usually generate detrimental effects. For example, directed evolution is like looking for a needle in a haystack, since billions of variants must be generated on genetic level, which are then functionally screened to eventually bring about a desired property. In this context, ncAA incorporation (especially our SPI-method) that endows proteins with chemical diversity that is not found in nature - is (at least form synthetic point of view) - a long awaited alternative technology with the potential to supplement and even replace these traditional approaches in biocatalyst design.  

Budisa, N. (2013). Expanded genetic code for the engineering of ribosomally synthetized and post-translationally modified peptide natural products (RiPPs)
Curr. Opin. Biotechnol. 24, 591-598.
Recombinant expression of antibiotics with unnatural/synthetic amino acids (in different combinations of their numbers and chemistry) will substantially diversify lantibiotics structure, leading to the creation of novel and unique sequence combinations, with some of these possessing  features important for human therapeutic use. Namely, peptide antibiotics are in the spotlight of many pharmaceutical companies because of they display high activities or specificities for target interaction, which is not limited to the field of antibacterial compounds. Immediately upon moving to Berlin, our group started to collaborate with Roderich Süssmuth on incorporation of various ncAAs in ribosomally synthesized lantibiotics
(10.1002/anie.201106154; 10.1002/cbic.201402558). We were the first to demonstrate that the physical properties of many ncAAs could directly influence the chemical and functional characteristics of lantibiotics, i.e. we achieved an additional level of chemical diversification of lantibiotic structures in bacterial hosts. This has great relevance for the development of new and unique active peptides with improved specificity, stability, membrane permeability, and with an improved oral availability. A further experiment on the co-translational incorporation of various unnatural/synthetic amino acids will certainly widely increase the number of possible chemical combinations in peptide-based antibiotic design.

Lepthien, S., Merkel, L., Budisa, N. (2010). In Vivo Double and Triple Labeling of Proteins Using Synthetic Amino Acids.
Angew. Chem. Int. Ed. Engl. 49, 5446-5450.
Initially we were working mainly on incorporation of various methionine and tryptophan analogues into proteins for protein structure elucidation by X-ray crystallography. Later we gradually extended this methodology to other research fields as well. In 2010 we achieved an important milestone in the development of our method: we reported the first successful simultaneous incorporation of three chemically distinct amino acids into one recombinant protein (in a single expression/incorporation experiment). In the following papers, we could even demonstrate the first efficient in vivo reassignment of up to four different in-frame sense codons (10.1039/C002256J) in one translation cycle or combination of sense/nonsense codon reassignments(10.1002/cbic.201000586). Such a methodology is not only a valuable toolkit for chemical biology but also for other research fields including protein chemistry and biophysics, cell biology, and pharmacology (more details see News from 2010 and 2011 on our web site).

Ma, Y., Biava, H., Contestabile, R., Budisa, N. & M.L. di Salvo. Coupling Bioorthogonal Chemistries with Artificial Metabolism: Intracellular Biosynthesis of Azidohomoalanine and its Incorporation into Recombinant Proteins.
Molecules, 2014, 19, 1004-1022.
Here we report that it is even possible to 'feed' bacteria toxic substances, such as azides, and produce useful ncAA Azidohomoalanine (Aha) intracellularly. This is important approach as it will enable us to design biotechnological processes for the production of high-quality protein-based molecules by the use of cheap and easily available starting materials. In the core of this novelty is a very efficient coupling of re-programmed translation with metabolic engineering, whereby a pathway for the biosynthesis of desired noncanonical amino acid can be engineered, imported and integrated into cellular metabolism. In this way, host cells would be able to generate a desired noncanonical amino acid from simple carbon sources and incorporate it directly in a high-value protein- or peptide-based product.  These are also important in the light of the fact that contemporary methodologies for ncAA-incorporations are well-suited for small-scale experiments focused on particular academic questions (e.g. florescence tags, pharmacological markers) but are not suitable for making robust artificial diversity in synthetic cells.

Bohlke N, Budisa N. (2014). Sense codon emancipation for proteome-wide incorporation of noncanonical amino acids: rare isoleucine codon AUA as a target for genetic code expansion.
FEMS Microbiol Lett., 351, 133-144.
Here we reported for the first time the "emancipation" of all 5797 rare AUA triplets from the canonical decoding in Escherichia coli (FEMS Microbiol Lett., 2014). This spectacular result provided a solid basis for the efficient incorporation of noncanonical amino acids as in contemporary experiments the efficiency of the method is much limited usually by out-competing the natural (canonical) amino acid substrates. It is believed that between 30 and 40 sense codons are enough to encode the genetic information of an organism - making a large number of sense codons (>20) available for recoding experiments with noncanonical amino acids. In this context, the genetic code should be substantially expanded by proteome-wide replacements with reassignments of sense codons - this would be a real step towards a synthetic organism (see also: link and link). Furthermore, sense-codon reassignment with noncanonical amino acids which will certainly circumvent all well-known pitfalls of suppression-based methods (low reproducibility, adverse context effects the read-through of stop-codons and non-triplet codons etc.). We have already published critical works (10.1371/journal.pone.003199210.1002/anie.201005680) regarding suppression-based methods in orthogonalized translation. In summary, the best way to circumvent the problem of the inefficiency of the current orthogonal translation is to focus on exploiting the degeneracy of the genetic code for the reassignment of rare codons. Until this is achieved, genetic code expansion will stay what it is now: an interesting extension of the classical recombinant DNA technology.

Hoesl, M.G., Oehm, S., Durkin, P., Darmon, E., Peil, L., Aerni, H-R., Rappsilber, J., Rinehart, J., Leach, D., Söll, D. & Budisa, N.  Chemical evolution of a bacterial proteome.
Angew. Chem. Int. Ed. Engl. 2015, 54, 10030-1003

We were first to report a long-term evolution which led to full 20,899 reassignments in the proteome of the bacterium E. coli. In particular, by quantitative substitution of tryptophan by suitable thienopyrrolyl-alanine analog, the chemical evolution of a synthetic bacterium is achieved. This breakthrough is a solid basis for design bacterial strains with altered genetic code that could lead to the creation of synthetic organisms is achieved. Contemporary methodologies for genetic code engineering are rather extensions of current recombinant DNA technology. They are well-suited for small-scale experiments focused on particular academic questions (e.g. florescence tags, pharmacological markers) but are not suitable for making robust artificial diversity in synthetic cells. In this context, the following is essential: the genetic code needs to be substantially expanded by proteome-wide replacements as this would be a real step towards a synthetic organism (for more details see News from 2015 on our web site).

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