Improving Co-Translational Incorporation of Non-Standard Amino Acids into Recombinant Proteins in Escherichia coliPublic Deposited
The genetic code, a set of rules by which mRNA codons are translated into the twenty standard amino acids used in protein synthesis, was once thought to be immutable. As we expanded our understanding of molecular biology more variations in this genetic code have been found across the animal kingdom. These findings have spurred interest in utilizing genetic code variations to incorporate non-standard, chemically diverse amino acids into proteins using the ribosome. By synthesizing proteins with novel chemical properties inside cells, we have the opportunity to transform how we synthesize materials and therapeutics, investigate protein structure, and understand the evolution of the translation system. The efficiency of non-standard amino acid (nsAA) incorporation still remains ~1000-fold lower than standard amino acid incorporation. This ultimately affects the total amount of modified protein possible to synthesize, limiting its application and use. In this thesis, I outline several strategies toward improving co-translational incorporation of nsAAs into recombinant proteins using Escherichia coli. The majority of my work has focused on developing an improved strain for nsAA incorporation. This has been accomplished by removing negative effectors of protein synthesis to increase modified protein yields up to 17-fold and by introducing a T7 system capable of tuning the expression of recombinant proteins. Additionally, I have demonstrated that independent promoter optimization and translational component evolution can be combined for a synergistic benefit. Specifically, we found that using an optimized promoter plasmid with engineered components resulted in a 2- to 20-fold enhancement of sfGFP expression containing multiple nsAAs. The work outlined here has furthered the development of co-translational incorporation of nsAAs into recombinant proteins and will help produce the next wave of highly functional biomaterials and protein therapeutics.
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