Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of translation termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before translation termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the translation dynamics of these two reporters allows visualization of a delay corresponding to the translation termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the translation termination rate. With CTELS, we systematically assessed negative effects of decreased 3′ UTR length, specifically on termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic translation system, mostly by affecting elongation, and that an excess of eRF1 termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a “leaky” stop codon context, which likely defines the basis of nonsense suppression.

Serum albumin (SA) plays a fundamental role in the transport of metabolites and endogenous ligands. Additionally, animal albumins are potent allergens. Heterologous expression of SAs is challenging due to their complex structure. In this study, we describe a simple method for enhanced production of soluble and functional recombinant feline (rFel d 2) and canine (rCan f 3) albumins in the standard E. coli strain BL21 (DE3). To achieve this, the 18-amino acid signal peptide was removed from the N-terminus of each albumin. To improve expression, folding, and solubility of recombinant proteins, we tested an extended panel of fusion proteins. Among them, MBP (maltose-binding protein), TF (trigger factor), and NusA (E. coli transcription termination factor) allowed the production of soluble fusion forms of rFel d 2 and rCan f 3. To confirm the structural integrity of the products, we analyzed the IgE-binding characteristics of recombinant versus native albumins. rFel d 2 and rCan f 3 fused with TF, MBP, and NusA bound albumin-specific immunoglobulin E (in 26/33, 29/33, 31/33, and 27/29, 26/29, 27/29 cases, respectively, comparable to native Fel d 2 and Can f 3. Thus, removal of the signal peptide combined with fusion partners enables expression of Fel d 2 and Can f 3 in E. coli with preserved antigenic properties.