Cell‐free translation systems for protein engineering

Abstract

Cell‐free translation systems have developed significantly over the last two decades and improvements in yield have resulted in their use for protein production in the laboratory. These systems have protein engineering applications, such as the production of proteins containing unnatural amino acids and development of proteins exhibiting novel functions. Recently, it has been suggested that cell‐free translation systems might be used as the fundamental basis for cell‐like systems. We review recent progress in the field of cell‐free translation systems and describe their use as tools for protein production and engineering.

Protein Engineering Methods and Applications

“…Rational design (Arnold, 1993) Site-directed mutagenesis (Arnold, 1993), (Antikainen & Martin, 2005) Evolutionary methods/directed evolution (Arnold, 1993) Random mutagenesis (Antikainen & Martin, 2005), (Wong et al, 2006), (Jackson et al, 2006), (Labrou, 2010) DNA shuffling (Antikainen & Martin, 2005), (Jackson et al, 2006) Molecular dynamics (Anthonsen et al, 1994) Homology modeling (Anthonsen et al, 1994) 'MolCraft'in vitro protein evolution systems (Shiba, 2004) Computational methods (computational protein design) (Jackson et al, 2006), (Van der Sloot et al, 2009), (Golynskiy & Seelig, 2010) Receptor-based QSAR methods (Lushington et al, 2007) NMR (Anthonsen et al, 1994) X-ray crystallography (Jackson et al, 2006) Peptidomimetics (Venkatesan & Kim, 2002) Phage display technology (Antikainen & Martin, 2005), (Sidhu & Koide, 2007), (Chaput et al, 2008) Cell surface display technology (Antikainen & Martin, 2005), (Gai & Wittrup, 2007), (Chaput et al, 2008) Flow cytometry / Cell sorting (Mattanovich & Borth, 2006 ) Cell-free translation systems (Shimizu et al, 2006) Designed divergent evolution (Yoshikuni & Keasling, 2007) Stimulus-responsive peptide systems (Chockalingam et al, 2007) Mechanical engineering of elastomeric proteins (Li, 2008) Engineering extracellular matrix variants (Carson & Barker, 2009) Traceless Staudinger ligation (Tam & Raines, 2009) De novo enzyme engineering (Golynskiy & Seelig, 2010) mRNA display …”

“…With further development, cellfree translation systems could be a strong alternative to in vivo protein expression, due to their high level of controllability and simplicity. The limitations of recombinant protein expression in living cells, such as protein degradation and aggregation will also be avoided (Shimizu et al, 2006).…”

Towards the engineering of in vitro systems

“…Undesired constituents of the cell-free extract might still interfere with system operation, or the precise composition of the system might vary as a consequence of prior cultivation conditions. For example, controlling all accessory protein components required for cell-free protein synthesis allows omitting release factor 1, which in turn allows direct recruitment of an amber stop codon for assignment of an additional amino acid (Shimizu et al 2006). Still, in particular, the cell-free extract approach was highly successful, most prominently in cell-free protein synthesis (Jewett et al 2008).…”

“…Of the two major applications for in vitro systems, cell-free protein synthesis (Shimizu et al 2006) and multi-enzyme reaction systems (Meyer et al 2007), this was attempted with some success for the former (Arnold et al 2005). A model to describe the cell-free production of GFP protein starting from gene expression from a T7 promoter could successfully predict (in some cases with rather good accuracy) quantitatively the behaviour of the concentration of a number of system components such as protein concentration, total mRNA concentration, acetyl phosphate concentration, adenosine phosphates and the levels of elongation factors Tu and T.…”

Integration of a Reconstituted Cell-free Protein-synthesis System on a Glass Microchip

“…A cell-free protein synthesis system 2,3 can meet the challenge of preparing multiple proteins. Such systems require only the addition of a template DNA or mRNA into the reaction mixtures and incubation for several hours to yield the proteins; they are therefore well suited for parallel syntheses of multiple proteins.…”