(569f) Accessing the Location-Dependent Effects of Unnatural Amino Acids on Protein Expression and Activity with Cell-Free Protein Synthesis-Facilitated Rapid Screening
AIChE Annual Meeting
2017
2017 Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Protein Engineering II: Combinatorial Techniques
Wednesday, November 1, 2017 - 2:00pm to 2:18pm
The incorporation of unnatural amino acids (uAA) can introduce novel functional groups into proteins with site-specific precision and control, with significant applications in basic science and protein engineering. However, uAA can decrease protein expression and functional activity depending on the location within the protein â a process that is not yet completely understood and difficult to predict. Therefore, practical applications often necessitate a time-consuming optimization of uAA location by individual expressions, purification and evaluations for many variant proteins. Such optimization can be accelerated through an in vitro system. We utilize this technology to simultaneously screen 24 different T4-Lysozyme mutants with different uAA incorporation sites in a matter of hours. Screening data offered a mechanistic explanation to some effects of uAA incorporation on expression and activity. Such effects include the solvent accessible surface area, the protein secondary structure, the distance from the active site, and the mRNA sequence. These effects are evaluated and their relative importance compare. In the end, structure-based guidelines to aid in the rational selection of uAA locations are presented. However, it remains challenging to predict the overall impact of uAA incorporation a prior, justifying the need for the above described rapid screen.
Citation:
Schinn SM, Bradley W, Groesbeck A, Wu JC, Broadbent A, Bundy BC. Rapid In Vitro Screening for the Location-Dependent Effects of Unnatural Amino Acids on Protein Expression and Activity. Biotechnology and Bioengeering. 2017. Apr 11. doi: 10.1002/bit.26305. Accepted.