Protein synthesis directly from PCR: progress and applications of cell-free protein synthesis with linear DNA

Abstract

A rapid, versatile method of protein expression and screening can greatly facilitate the future development of therapeutic biologics, proteomic drug targets and biocatalysts. An attractive candidate is cell-free protein synthesis (CFPS), a cell-lysate-based in vitro expression system, which can utilize linear DNA as expression templates, bypassing time-consuming cloning steps of plasmid-based methods. Traditionally, such linear DNA expression templates (LET) have been vulnerable to degradation by nucleases present in the cell lysate, leading to lower yields. This challenge has been significantly addressed in the recent past, propelling LET-based CFPS as a useful tool for studying, screening and engineering proteins in a high-throughput manner. Currently, LET-based CFPS has promise in fields such as functional proteomics, protein microarrays, and the optimization of complex biological systems.

Escherichia coli-based cell-free extract development for protein-based cancer therapeutic production

“…A combination of this modified cell-free method and linear template DNA, which has been studied and engineered by a number of researchers (He and Taussig, 2001, Woodrow et al, 2006, Yang et al, 1980, could facilitate rapid screening for a mutant onconase with increased potency against cancer cells. More information about using linear DNA templates and high-throughput CFPS can be found in recent reviews and papers (Murray and Baliga, 2013, Schinn et al, 2016, Yabuki et al, 2007.…”

“…• Open Nature  Ease of monitoring (Smith et al, 2014b)  Ease of optimization (Smith et al, 2014b)  No transport limitation (Swartz, 2012) • Higher toxicity tolerance (Casteleijn et al, 2013) • Ease of Manipulation  uAA incorporation (Zimmerman et al, 2014)  Disulfide bond formation Swartz, 2011, Goerke and  Folding using chaperones (Spirin and Swartz, 2008)  Addition of exogenous materials (Swartz, 2012) • Fast protein synthesis (Zawada et al, 2011) • Ability to use linear DNA template (Schinn et al, 2016) • High-throughput screening (Woodrow et al, 2006, Woodrow and • Scalable (Zawada et al, 2011) • Platform for personalized medicines (Kanter et al, 2007) • Lyophilization capable  Ease of stockpiling reagents (Smith et al, 2014a)  Ease of transportation (Smith et al, 2014a) • Sterilization method (Smith et al, 2015) • Not yet an FDA-approved Process • Higher reagent cost compared to in vivo systems (Swartz, 2006) • Largest industrial scale fermentation demonstrated is 100 L (Zawada et al, 2011) • Limited protein synthesis life time (typically 3-12 hours in batch format) (Michel-Rydellet et al, 2004) • Lack of cell-free specific bioreactor development reported in literatura…”

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

“…The linear template degradation which is problematic without substantial intervention, is fairly common in cell‐free systems, as well (Jahanban‐Esfahlan et al, 2017; Seidi et al, 2018). However, LET‐based CFPS is a practical instrument for high‐throughput studies and screenings of engineered proteins (Schinn, Broadbent, Bradley, & Bundy, 2016).…”

Efficient tRNA degradation and quantification in Escherichia coli cell extract using RNase‐coated magnetic beads: A key step toward codon emancipation

“…A minimized genetic code promises to expand the available proteomic toolset with implications in many fields, including biotherapeutics, biocatalysis, protein labeling, and minimal cells . Such a rewriting of the genetic code requires the decoupling and reengineering of the codon–tRNA–amino acid relationships, which defines the genetic coding of proteins by nucleic acids .…”