Cell-free Platforms for Synthesis of Non-standard Polypeptides in vitro

Des Soye, Benjamin James

doi:https://doi.org/10.21985/n2-xae4-v421

Work

Cell-free Platforms for Synthesis of Non-standard Polypeptides in vitro

Public

Download PDF

Download All Files (.zip)

Proteins represent a critical class of biomolecules, universally employed by all living organisms to fulfill essential structural, functional, and enzymatic roles necessary to support life. In nature, these polymers are composed generally of twenty natural amino acid (AA) building blocks, which can be modified with covalent adducts known as post-translational modifications (PTMs) to effect changes in functionality and behavior. Complete understanding of the biological role played by a given protein necessitates an understanding of its various PTMs and their influence on its structure and function. However, our ability to identify and characterize specific patterns of PTMs is limited by our ability to produce pure, homogeneous samples of proteins featuring a specific set of modifications. In this thesis, I sought to develop platforms for enabling preparative scale synthesis of proteins featuring user-definable PTM patterns to facilitate downstream fundamental discovery. The key idea was to genetically encode PTMs and then directly incorporate modified amino acids (as non-canonical amino acids, ncAAs) into proteins by repurposing the amber stop codon as a coding channel, a technique known as amber suppression. Unfortunately, up to now these approaches have been conventionally limited by competition from release factor 1 (RF1) and the fact that the orthogonal translation systems (OTSs) used to incorporate ncAAs are toxic to cells. To address these limitations, I hypothesized that applying cell-free protein synthesis (CFPS) systems derived from cells lacking RF1 would enable the use of OTSs without associated toxicity effects while simultaneously eliminating release factor competition. I further hypothesized that the elimination of putative negative effectors of CFPS or the use of highly-active translational components would yield highly-productive CFPS systems, enabling preparative scale synthesis of proteins featuring specific PTMs for downstream characterization and thus allowing me to meet my goal. I first contributed to the development of a CFPS system from a partially-recoded strain of Escherichia coli deficient RF1. As hypothesized, stabilization of template DNA and mRNA via removal of nucleases in the system increased productivity fourfold, and ncAA incorporation was improved significantly in the absence of RF1. Next, I embarked on a similar effort to generate a CFPS system from a fully-recoded RF1-deficient strain in which all native instances of the amber stop codon were removed. In further agreement with my initial hypotheses, the functional inactivation of putative negative effectors of CFPS yielded a 4.5-fold increase in platform productivity and the absence of RF1 activity in the system facilitated insertion of 40 identical ncAAs into proteins with ≥98% fidelity. In my next aim, I hypothesized that imbuing our best recoded RF1-deficient source strain with the ability to synthesize the viral T7 RNA polymerase would expand the capabilities of our recoded CFPS platform to yield a one-pot system, simplifying use of the platform and facilitating its adoption. After genomically incorporating a construct encoding the polymerase into the strain and subsequently installing mutations protecting the enzyme from proteolysis, the platform was capable of robust ncAA incorporation independent of purified polymerase supplementation, agreeing with my hypothesis. Next, I set out to pioneer a novel CFPS system derived from the fast-growing non-model bacterium Vibrio natriegens based on the hypothesis that its lysates would be enriched with highly-active translational components. Elucidation of optimal cell growth, lysis, and reaction conditions culminated in a highly-productive CFPS platform comparable to the state-of-the-art in agreement with this hypothesis. I also found that the system was uniquely capable of synthesizing short peptides, which have historically been difficult to produce recombinantly. Finally, I pursued the synthesis of proteins featuring specific patterns of serine phosphorylation, a widely studied PTM. In this effort, I reasoned that incorporation of o-phosphoserine (Sep) in CFPS could be improved by utilizing improved Sep-specific translation components in an engineered strain background optimized for Sep incorporation. In an illustrative example of what I was trying to accomplish, I applied our systems to the synthesis of the glycolytic enzyme triosephosphate isomerase bearing specific serine phosphorylations to investigate their effects on the enzyme. Taken together, my work will facilitate efforts to interrogate the effects of specific PTMs on protein structure and function, further increasing our understanding of the chemistry of life.

Creator