Keywords

low-cost cell-free uAA incorporation, alternative energy systems for CFPS, LET-based rapid gene library screening

Abstract

Site-specific incorporation of unnatural amino acids (uAAs) during protein synthesis expands the proteomic code through the addition of unique residue chemistry. This field provides a unique tool to improve pharmacokinetics, cancer treatments, vaccine development, proteomics and protein engineering. The limited ability to predict the characteristics of proteins with uAA-incorporation creates a need for a low-cost system with the potential for rapid screening. Escherichia coli-based cell-free protein synthesis is a compelling platform for uAA incorporation due to the open and accessible nature of the reaction environment. However, typical cell-free systems can be expensive due to the high cost of energizing reagents. By employing alternative energy sources, we reduce the cost of uAA-incorporation in CFPS by 55%. While alternative energy systems reduce cost, the time investment to develop gene libraries can remain cumbersome. Cell-free systems allow the direct use of PCR products known as linear expression templates, thus alleviating tedious plasmid library preparations steps. We report the specific costs of CFPS with uAA incorporation, demonstrate that LETs are suitable expression templates with uAA-incorporation, and consider the substantial reduction in labor intensity using LET-based expression for CFPS uAA incorporation.

Original Publication Citation

Prashanta Shrestha, Mark Thomas Smith, Bradley Charles Bundy, Cell-free unnatural amino acid incorporation with alternative energy systems and linear expression templates, New Biotechnology, Volume 31, Issue 1, 2014, Pages 28-34, ISSN 1871-6784, https://doi.org/10.1016/j.nbt.2013.09.002.

Document Type

Peer-Reviewed Article

Publication Date

2013-10-06

Publisher

New Biotechnology

Language

English

College

Ira A. Fulton College of Engineering

Department

Chemical Engineering

University Standing at Time of Publication

Full Professor

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