433589 Rapid, High-Throughput Screening of Proteins with Non-Canonical Residues Using Cell-Free Protein Synthesis

Tuesday, November 10, 2015: 8:50 AM
150G (Salt Palace Convention Center)
Song-Min Schinn1, Steven K. Stanley1, Ashtyn Groesbeck1, Kristen M. Wilding1, Jeffrey C. Wu2 and Bradley C. Bundy1, (1)Chemical Engineering, Brigham Young University, Provo, UT, (2)Department of Chemical Engineering, Department of Molecular Biology, Brigham Young University, Provo, UT

The site-specific incorporation of non-canonical amino acids (ncAA) can enhance protein characteristics towards improved therapeutics, medical diagnostics and biocatalysts. However, predicting the characteristics of proteins with such novel residues is still difficult. Simulation methods, which often depend heavily on homology, are ill-suited to deal with proteins containing novel residues. Conventional in vivo screening methods require a plasmid DNA library, whose cloning-based techniques are time- and labor-intensive. Therefore, there is an unmet need for a rapid, high-throughput screening system to probe the characteristics of proteins with ncAA residues.

Cell-free protein synthesis is an attractive alternative platform for ncAA incorporation due to the open and accessible nature of the reaction environment. Further, cell-free systems can express proteins from PCR-produced linear templates, and thus can avoid cumbersome cloning steps. Here, we engineer a screening technique that coupling PCR-based library generation and cell-free protein synthesis.  Unique ncAA incorporation sites in a model biocatalyst were screened in parallel to optimize enzymatic activity and functional stability, while significantly reducing labor time compared to popular in vivo methods.

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See more of this Session: Biomolecular Engineering
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division