A new cell-free platform for effective site-specific incorporation of non-natural amino acids and screening for orthogonal components [electronic resource]
Keywords: Chemical Engineering

Proteins in all living systems are composed predominantly of 20 natural amino acids, each of which is incorporated by a processive mechanism involving dedicated transfer RNAs and enzymes called aminoacyl-tRNA synthetases. Despite the diversity in protein structure and function, these natural amino acids provide side chains with only limited reactivity. Site-specific incorporation of non-natural amino acids (nnAAs) expands the chemical reactivity of proteins and enables their precise post-translational modification. These nnAAs are incorporated in a manner analogous to natural amino acids. An orthogonal synthetase specific to the nnAA first couples the nnAA to an orthogonal amber suppressor tRNA (o-tRNA). The aminoacylated o-tRNA then forms a three-molecule complex with Ef-Tu and GTP, enters the ribosome, and at an amber stop codon (UAG) in the mRNA, adds the nnAA to the nascent polypeptide chain. Of the more than 30 nnAAs that have been site-specifically incorporated using Escherichia coli-based protein synthesis systems, the Swartz laboratory has been particularly interested in the nnAAs p-azido-L-phenylalanine (pAzF) and p-propargyloxy-L-phenylalanine (pPaF), since proteins containing these nnAAs can be directly coupled using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. In our laboratory, this technology is used to design unique and effective vaccines and diagnostic agents. Despite the improvements in the versatility and productivity of the cell-free protein synthesis (CFPS) platform, the yields of proteins containing nnAAs (i.e. modified proteins) have been substantially lower than their natural counterparts. The first part of this dissertation summarizes our efforts in improving the current methods for site-specific nnAA incorporation. Supplementing the initial CFPS system with additional o-tRNA doubled the modified protein yields. Titration experiments and turnover calculations suggested that, despite the apparent o-tRNA limitation, the synthetase is the more inefficient orthogonal component. Nonetheless, a convenient and modular method for adequate o-tRNA supply was demonstrated to further improve the efficiency of nnAA incorporation. Using this new method, in which the o-tRNA and the modified protein are produced simultaneously during the CFPS reaction, as much as 1.67 mg/ml full-length and soluble modified super-folder GFP (sfGFP) was obtained. This represents a 6-fold improvement over previous cell-free yields of modified sfGFP. This new method was used then to study nnAA incorporation at twelve different sites in sfGFP using two different o-tRNA sequences. Our results did not confirm the previous trends for the position effect (i.e. variation of modified protein yield with nnAA incorporation position). The use of the o-tRNA that was developed to better recognize the endogenous Ef-Tu decreased the size of the position effect. In addition, the method employing in situ o-tRNA synthesis was extended to the incorporation of the nnAAs into sfGFP at 2 or 3 positions. These proteins were subsequently purified and coupled to synthesize linear and branched protein polymers. After polymerization and removal of the catalyst, the specific activity of the proteins was fully retained. The second part of this dissertation describes our efforts towards developing a new set of orthogonal components from the endogenous tyrosyl tRNA/synthetase pair. A cell-free method was first developed to screen for an orthogonal variant of the E. coli tyrosyl tRNA. 201 potential orthogonal tRNAs were identified out of a library containing 384 tRNA mutants. Eight were more carefully analyzed for orthogonality (i.e. absence of cross-reactivity with E. coli synthetases) and one was identified to be both sufficiently orthogonal and not inhibitory to CFPS. Finally, the in vitro compartmentalization (IVC) method was adapted for the production of sfGFP in emulsion CFPS reactions. A method was also developed to quantify the number of adsorbed genes on a bead by fluorescence using FACS. This protocol promises to be a powerful high-throughput method for screening orthogonal synthetases.