Robotic Chemical Protein Synthesis for the Experimental Validation of the Functional Annotation of Microbial Genomes

Modern total protein synthesis has evolved from the ‘chemical ligation’ methods introduced by the Kent laboratory [Kent SBH. Total chemical synthesis of proteins. Chemical Society Reviews 2009; 38: 338-51.]. Unprotected synthetic peptide segments, spanning the amino acid sequence of the mature polypeptide chain derived from a predicted open reading frame, are covalently joined to one another by chemo-selective reaction. Native chemical ligation, the thioester-mediated covalent bond-forming chemoselective reaction of unprotected peptides at a Cys residue, is the most robust and useful ligation chemistry developed to date. The synthetic protein is then used to experimentally validate the predicted biochemical function, and in selected cases to determine the Xray structure of the protein molecule (Figure). Figure 1. Modular high-throughput platform for fast and parallel total chemical synthesis, mass-spectrometric purification and single-molecule spectroscopic assay to annotate function for newly predicted proteins.