In vitro synthesis of 32 translation-factor proteins from a single template reveals impaired ribosomal processivity

Abstract

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.

Min waves without MinC can pattern FtsA-FtsZ filaments on model membranes

“…The expression lifespan, defined as the time point at which protein production stops, was determined for each protein: 119 ± 28 min (FtsA), 144 ± 28 min (MinD) and 250 ± 16 min (MinE). These values are consistent with those obtained from kinetic profiles of other PUREexpressed proteins 34,35 .…”

Biochemistry of Aminoacyl tRNA Synthetase and tRNAs and Their Engineering for Cell-Free and Synthetic Cell Applications

“…In a similar attempt, all 20 AARSs were expressed using a polycistronic plasmid encoding 32 proteins in total using the PURE system. The PURE system was able to synthesize all 32 proteins, including all 20 AARSs as confirmed by mass spectrometric analysis but functionality was not assessed (Doerr et al, 2021). Recently, in the bid to construct a self-replicating synthetic cell, a modified PURE system was used to demonstrate self-regeneration of up to 7 AARSs in a microfluidic reactor for more than 24 h (Lavickova, Laohakunakorn, and Maerkl, 2020).…”

Shaping Liposomes by Cell-Free Expressed Bacterial Microtubules

“…We chose the PURE system, a reconstituted E. coli -based translation machinery (specifically the commercially available PURE frex 2.0), as our CFPS platform. This choice was motivated by the very low levels of protease and nuclease activity, and the wide range of (membrane) proteins synthesized in an active state with this system. ,,− DNA templates for PURE frex 2.0 reactions consisted of the btubA and btubB genes from Prosthecobacter dejongeii . Both DNA constructs were sequence-optimized for (i) expression in an E. coli host by matching codon occurrence with tRNA usage, (ii) low guanine−cytosine (GC) content within the first 30 base pairs (synonymous mutations were introduced to keep the amino acid sequence unaltered), and (iii) a low propensity of intramolecular base pairing of the mRNA around (in the vicinity or involving) the start codon (Figure S1).…”