The Protein synthesis Using Recombinant Elements (PURE) system enables transcription and translation of a DNA template from purified components. Therefore, the PURE system-catalyzed generation of RNAs and proteins constituting the PURE system itself represents a major challenge toward a self-replicating minimal cell. In this work, we show that all translation factors (except elongation factor Tu) and 20 aminoacyl-tRNA synthetases can be expressed in the PURE system from a single plasmid encoding 32 proteins in 30 cistrons. Cell-free synthesis of all 32 proteins is confirmed by quantitative mass spectrometry-based proteomic analysis using isotopically labeled amino acids. We find that a significant fraction of the gene products consists of proteins missing their C-terminal ends. The per-codon processivity loss that we measure lies between 1.3 × 10–3 and 13.2 × 10–3, depending on the expression conditions, the version of the PURE system, and the coding sequence. These values are 5 to 50 times higher than those measured in vivo in E. coli. With such an impaired processivity, a considerable fraction of the biosynthesis capacity of the PURE system is wasted, posing an unforeseen challenge toward the development of a self-regenerating PURE system.

Trypsin digest: Per LC-MS injection, 1.5 µL of PURE system reaction was mixed with 3 µL of 100 mM Tris-HCl pH8.0, 0.3 µL of 20 mM CaCl2, and 0.975 µL MilliQ water. Samples were incubated at 90 °C for 10 min and after cooling to room temperature 0.225 µL of 1 mg mL–1 trypsin (trypsin-ultra, MS-grade, New England Biolabs) was added. Samples were then incubated at 37 °C overnight. After addition of 0.6 µL 10% trifluoroacetic acid samples were centrifuged in a table-top centrifuge (5415R, Eppendorf) for 10 min at maximum speed. The supernatant was transferred to a glass vial with small-volume insert for LC-MS/MS analysis. For absolute quantitative proteomic analysis three different concentrations of PUREfrex2.0 and PURExpress samples were mixed with a fixed concentration of both QconCAT halves. Samples were digested with trypsin as described above and, before LC-MS/MS analysis, they were supplemented with 110 nM of 13C-Arg/Lys labeled SILs (Pepscan presto, Lelystad, The Netherlands) corresponding to the two quantification peptides on the QconCAT halves. Proteomic analysis: LC-MS/MS analysis was performed on a 6460 Triple Quad LCMS system (Agilent Technologies, USA) using Skyline software. 5.5 µL of sample was injected per run to an ACQUITY UPLC® Peptide CSH™ C18 Column (Waters Corporation, USA). The peptides were separated in a gradient of buffer A (25 mM formic acid in MilliQ water) and buffer B (50 mM formic acid in acetonitrile) at a flow rate of 500 µL per minute and at a column temperature of 40 °C. The column was equilibrated with 98% A. After injection, the gradient was changed linearly over 20 min to 70% buffer A, over the next 4 min to 60% buffer A, and over the next 30 s to 20% buffer A. This ratio was held for another 30 s and the column was finally flushed with 98% buffer A to equilibrate for the next run. Selected peptides were measured by multiple reaction monitoring (MRM). For reactions with expression of pTFM1 measurements were split over three LC-MS/MS runs. For reactions including 15N-labeled amino acids, transitions for peptides containing 15N-amino acids were monitored, except for glutamate because of the excess of the light glutamate contained in the buffer.

The sample is consisting of PURE cell free expression system obtained from GeneFrontier (japan). Purified DNA with T7 or SP6 promotor can be readily expressed in the system and in solution digestion with trypsin can be conducted without a a problem.