Development of a Non-Radioactive Aminoacylation Assay for Quality Control of Purified and Reconstituted In Vitro Translation Systems

Abstract

In order to understand how proteins folding during translation on the ribosome, we require efficient and highly controllable in-vitro translation systems capable of faithfully synthesizing nascent protein chains. Purified and reconstituted in vitro translation (e.g. PURE IVT) systems meet this need. However, to ensure high-fidelity translation the numerous activities involved must each be properly characterized prior to reconstitution of full-scale translation. In this study, we focused on improving a non-radioactive spectrophotometric assay that facilitates the determination of aminoacyl-tRNA synthetase activities. This assay, monitors pyrophosphate/phosphate generation as a proxy for aminoacylation and can thus be used to monitor the activities of all 20 amino-acyl tRNA synthetase enzymes required for translation. Phosphate generation is coupled to a spectrophotometric change using purine nucleoside phosphorylase (PNPase) which catalyzes phosphorylation of MESG substrates (Amax∼330nm) to make AMMP (Amax∼360nm) products. The main objective of the present study was to clone, express, and purify highly purified (i.e. phosphate-free and ATPase-free) PNPase for the determination of PURE IVT system component activities. This assay will enable the non-radioactive characterization of the activities of natural as well as engineered aminoacyl-tRNA synthetases as required for high-fidelity unnatural amino acid incorporation for co-translational protein folding studies.