Cell-free protein synthesis: the state of the art

Abstract

Cell-free protein synthesis harnesses the synthetic power of biology, programming the ribosomal translational machinery of the cell to create macromolecular products. Like PCR, which uses cellular replication machinery to create a DNA amplifier, cell-free protein synthesis is emerging as a transformative technology with broad applications in protein engineering, biopharmaceutical development, and post-genomic research. By breaking free from the constraints of cell-based systems, it takes the next step towards synthetic biology. Recent advances in reconstituted cell-free protein synthesis (Protein synthesis Using Recombinant Elements expression systems) are creating new opportunities to tailor the reactions for specialized applications including in vitro protein evolution, printing protein microarrays, isotopic labeling, and incorporating nonnatural amino acids.

Figure 1

PURE protein translation system. The PURE expression system is programmed by a DNA template and includes a transcription module, an energy-coupling module for NTP regeneration, aminoacyl tRNA synthetases and tRNAs, and a translation module comprised of ribosomes, elongation factors, and release factors that are integrated to perform peptide elongation and macromolecular synthesis.

Figure 2

ATP regeneration pathways for cell-free protein synthesis. Commonly used reaction sequences (Cre-P (Creatine-phosphate); PANOxSP (Phosphoenolpyruvate (PEP), Amino acids, NAD⁺, Oxalic acid, Spermidine and Putrescine)) are indicated. The enzymes required for these reactions are: (1) Creatine kinase; (2) hexokinase; (3) phosphofructokinase; (4) aldolase + triose phosphate isomerase + glyceraldehydes-3-phosphate dehydrogenase + 3-phosphoglycerate mutase + enolase; (5) pyruvate kinase; (6) pyruvate dehydrogenase; (7) phosphate acetyltransferase ; (8) acetate kinase; (9) lactate dehydrogenase; (10) pyruvate oxidase; (11) phosphorylase; (12) phosphoglucomutase.