High-throughput engineering of proteins: Sampling extended chemical diversity by combining directed evolution with an expanded genetic code.

Every living thing contains thousands of different proteins that perform most of the crucial jobs needed to maintain life. Proteins are also being utilised outside of their normal biological context as therapeutics in industry. Proteins are molecules that consist of repeating units of chemicals called amino acids. In the vast majority of cases, only 20 different amino acids are used to compose a single protein. This means that the chemical nature of proteins is restricted to what is available from the 20 amino acids. This in turn limits the available functionality that a protein can sample. Expanding the type of chemistry available to a protein will allow new properties not currently available in nature to be introduced, which will open up new routes to the study of biological systems and for adapting proteins for biotechnological applications. Therefore, we hypothesise that expanding the chemical diversity sampled by a protein by the incorporation of unnatural amino acids can introduce new chemistry into a protein so generating variants with novel and enhanced properties. The sequence of amino acids that comprise a protein is encoded in the genetic material via a gene. One approach for incorporation of unnatural amino acids into a protein within a cell is to alter the coding properties of a gene via changes to the individual codons comprising the gene (codons encode precisely which amino acid will be added to the growing protein chain). This can be achieved by altering or engineering components of the cell protein synthesising machinery so that the unnatural amino acid is incorporated into the growing protein chain in response to a specific codon. In this project, we present a method to genetically encode any number of novel unnatural amino acids into any protein. Our method targets the entire sequence of a protein in a high-throughput manner such that potentially every possible amino acid position is examined for its effects on the properties of a protein. This approach, termed directed evolution, is a powerful and successful strategy based on Darwinian evolution to alter the properties of proteins but is targeted to an individual gene and takes place in a controlled fashion in the test-tube. Our proposed method will be the first to demonstrate that artificial protein evolution can involve the use of unnatural synthetic amino acids. Since our knowledge of the effect of incorporating an unnatural amino acid at particular amino acid position in a protein is limited, having a method that can quickly sample many different positions throughout a protein would be extremely useful. This will in turn allow us to fully exploit the potential of constructing proteins with new and useful properties through in the sampling of new chemical functionalities.

The project aims to use a new combined protein evolution and synthetic biology strategy to widen the chemical and hence functional diversity sampled by proteins through the use of an expanded genetic code. Unnatural amino acids have a rich diversity of physicochemical properties not present in any of the common 20 amino acids. Expansion of the genetic code to allow the precise incorporation of such unnatural amino acids during cellular protein synthesis opens up the possibility of efficiently producing proteins with novel chemical, physical and biological properties not normally accessible in nature. Given our limited knowledge concerning the influence of unnatural amino acids on protein structure, it is difficult to predict the consequence of substituting a natural amino acid for an unnatural one at any given residue position. Therefore, a new protein mutagenesis method to be used in conjunction with expanded genetic code systems will be developed and implemented to broaden the chemical space sampled by proteins. This new and generally applicable directed evolution method will replace single codons randomly throughout a target gene with new codons specifying incorporation of an unnatural amino acid. Initially, genes encoding TEM-1 beta-lactamase and enhanced GFP will be the targets. Engineered orthogonal tRNA/aminoacyl-tRNA synthetase pairs will be used to incorporate a desired unnatural amino acid into the growing polypeptide in vivo in response to a designated codon sequence, such as the amber nonsense codon (UAG). The codon replacement gene library will then be screened to identify variants and hence residue positions that tolerate the incorporation of various different unnatural amino acids. Finally, the influence of the unnatural amino acid and specific post-translational modification of the unnatural amino acid side-chain, such a PEGylation, on protein function and stability will be investigated in vivo and in vitro.