Fig. 2: Design and evaluation of expression cassette. | Nature Communications

Fig. 2: Design and evaluation of expression cassette.

From: Towards a generic prototyping approach for therapeutically-relevant peptides and proteins in a cell-free translation system

Fig. 2: Design and evaluation of expression cassette.

a Design of expression construct: universal initiation sequence (SITS) comprising the unstructured 5′UTR, start-codon (ATG) and 3′ part, encoding for the leader peptide (green), directs translation of peptide-of-interest (POI) carrying the RGS-tag (yellow). A TVMV-cleavage site (pink) is included for co-translational removal of the SITS-derived translation leader. b Comparative performance of different translation initiation sequences including full SITS, classical Shine-Dalgarno Ribosome Binding Site (SD-RBS) and unstructured 5′UTR derived from SITS (5′-SITS) in supporting the synthesis of peptides from different size classes such as 8 aa RGS peptide (RGS), RGS fusion with 14 aa SFTI (SFTI-RGS) and 57 aa Dc1a (Dc1a-RGS) (Table 1). Peptides were quantified using affinity-clamp assay and initial rates of substrate cleavage are plotted as bars or dots for peptide variants produced in S30-based (S30) or PURE (Protein synthesis Using Recombinant Elements) translation systems, respectively. The results are represented as means ± s.d. of n = 3 repeats of the assay.

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