Abstract
Incorporating non-standard amino acids (NSAAs) into proteins enables new chemical properties, new structures, and new functions. In recent years, improvements in cell-free protein synthesis (CFPS) systems have opened the way to accurate and efficient incorporation of NSAAs into proteins. The driving force behind this development has been three-fold. First, a technical renaissance has enabled high-yielding (>1 g/L) and long-lasting (>10 h in batch operation) CFPS in systems derived from Escherichia coli. Second, the efficiency of orthogonal translation systems (OTSs) has improved. Third, the open nature of the CFPS platform has brought about an unprecedented level of control and freedom of design. Here, we review recent developments in CFPS platforms designed to precisely incorporate NSAAs. In the coming years, we anticipate that CFPS systems will impact efforts to elucidate structure/function relationships of proteins and to make biomaterials and sequence-defined biopolymers for medical and industrial applications.
Highlights
The incorporation of non-standard amino acids (NSAAs) into proteins andpeptide-based materials is a key emerging application area in synthetic biology (Liu and Schultz, 2010; Hoesl and Budisa, 2012)
Efforts to incorporate NSAAs using cell-free protein synthesis (CFPS) systems based on Escherichia coli have grown significantly
Recent advances have addressed some of these challenges by improving NSAA incorporation efficiency by engineering o-transfer RNA (tRNA) (Young et al, 2010; Chatterjee et al, 2012), o-aaRS (Liu et al, 1997; Chatterjee et al, 2012), or Elongation Factor Tu (EF-Tu) (Doi et al, 2007; Park et al, 2011) as well as controlling transcription and translation rate (Young et al, 2010; Chatterjee et al, 2013), and removing release factor 1 (RF1) competition (Mukai et al, 2010; Johnson et al, 2011; Loscha et al, 2012; Lajoie et al, 2013)
Summary
The incorporation of non-standard amino acids (NSAAs) into proteins and (poly)peptide-based materials is a key emerging application area in synthetic biology (Liu and Schultz, 2010; Hoesl and Budisa, 2012). Efforts to incorporate NSAAs using cell-free protein synthesis (CFPS) systems based on Escherichia coli have grown significantly. We describe CFPS and recent improvements in NSAA incorporation in crude cell extract as well as reconstituted systems of purified components.
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