| Recent developments have made E. coli-based cell-free systems a viable alternative to conventional recombinant DNA expression technologies. Such advances of the cell-free technology provide exciting new opportunities to develop innovative and effective therapeutics and vaccines that are time consuming and costly to produce with traditional technologies.; This dissertation begins by describing an assessment of modern cell-free methods. As a result, cell-free platforms were established to produce nine complex proteins. Reduction in raw material costs was achieved by developing a cell-free system that produces disulfide bonded proteins while using glucose as an energy source. Benefits of such cell-free systems are highlighted when producing a luciferase that requires five disulfide bonds. Our cell-free system produced Gaussia luciferase in high yields and with activity greater than any characterized luciferase.; To enable targeted post-translational modification of proteins produced in our cell-free technology, a new system was developed to produce proteins with non-natural amino acids (nnAA) incorporated site-specifically. The mutant Methanococcus jannaschii tyrosyl-tRNA synthetase and tRNATyr pair were used as orthogonal elements. The three nnAAs, O-methyl-L-tyrosine, p-acetyl-L-phenylalanine, and p-azido-L-phenylalanine (pAz) were incorporated using the new cell-free system. Effective nnAA incorporation resulted in unprecedented cell-free yields of modified protein.; We extended this technology to efficiently incorporate nnAAs into disulfide bonded proteins, vesicle-integrated membrane proteins, and virus-like particles (VLPs). The aim was to conduct unique attachment chemistries on protein surfaces. Protein surface modification required the development of a simplified yet improved (3+2) cycloaddition reaction environment that retained the disulfide bonds necessary for protein bioactivity. Following cycloaddition reaction optimization, the attachment efficiency of an alkynyl-PEG chain to pAz was nearly 100% while maintaining the correctly formed disulfide bonds. Additionally, an azide-reactive fluorophore was attached to membrane proteins and alkynyl-PEG conjugated to the VLP surface.; Lastly, unique linkers were synthesized and used to create protein assemblies. This work is the first report of using nnAAs to successfully conjugate disulfide bonded proteins as well as to attach a disulfide bonded protein to the surface of a VLP. When combined, this technology enables the design and production of custom-designed drug delivery vehicles and vaccines with unprecedented versatility. |
