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. 2017 Apr 24:7:46753.
doi: 10.1038/srep46753.

A Novel Method to Evaluate Ribosomal Performance in Cell-Free Protein Synthesis Systems

Noémie Kempf  1 , Cristina Remes  1 , Ralph Ledesch  1 , Tina Züchner  1 , Henning Höfig  1   2 , Ilona Ritter  1 , Alexandros Katranidis  1 , Jörg Fitter  1   2
Affiliations

A Novel Method to Evaluate Ribosomal Performance in Cell-Free Protein Synthesis Systems

Noémie Kempf et al. Sci Rep. .

Abstract

Cell-free protein synthesis (CFPS) systems were designed to produce proteins with a minimal set of purified components, thus offering the possibility to follow translation as well as protein folding. In order to characterize the performance of the ribosomes in such a system, it is crucial to separately quantify the two main components of productivity, namely the fraction of active ribosomes and the number of synthesizing cycles. Here, we provide a direct and highly reliable measure of ribosomal activity in any given CFPS system, introducing an enhanced-arrest peptide variant. We observe an almost complete stalling of ribosomes that produce GFPem (~95%), as determined by common centrifugation techniques and fluorescence correlation spectroscopy (FCS). Moreover, we thoroughly study the effect of different ribosomal modifications independently on activity and number of synthesizing cycles. Finally, employing two-colour coincidence detection and two-colour colocalisation microscopy, we demonstrate real-time access to key productivity parameters with minimal sample consumption on a single ribosome level.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Cartoon of the constructs used.
The gene of GFPem was followed by a 30-residue linker rich in Gly and Ser, the 17-residue SecM AP or SecMstr AP and a stop codon. The mutated amino acids of SecMstr AP are shown in red letters. The control construct is missing the last 14 residues of the SecM moiety. All constructs could be linearized removing at the same time the stop codon.
Figure 2
Figure 2. Stalling efficiency of different constructs as determined by co-precipitation.
(a) Relative amount of GFPem (after 2 h CFPS reaction) in supernatant (cyan background) and pellet (red background) after centrifugation through sucrose for circular (light blue bars) and linear (dark blue bars) constructs. High amounts of GFPem in the supernatant (No SecM, SecM) suggest substantial release of the nascent chain. On the contrary, high amounts in the pellet (SecMstr) indicate efficient stalling. The experiments were carried out at least 3 times for each condition (biological replications). (b) Corrected values of diffusion coefficients D0 (see Supplementary Fig. S1) measured in FCS for formula image and formula image without centrifugation (after 2 h CFPS reaction). Corresponding values for free GFPem and labelled 70S ribosomes are given for comparison. The intermediate value for formula image reveals partial release, while the ribosome-like diffusion of formula image indicates efficient stalling.
Figure 3
Figure 3. Activity and number of synthesizing cycles for different ribosomal modifications.
(from left to right) PURE System ribosomes; CAN20/12E ribosomes; biotinylated CAN20/12E ribosomes; biotinylated CAN20/12E ribosomes labelled with Cy5. Activity (red bars) and number of productive cycles (blue bars) were determined by expressing and measuring the concentration of formula image (efficient stalling, only one productive cycle) and formula image (almost no stalling, constant activity assumed), respectively (after 2 h CFPS reaction). Activity drops with each modification step, while the number of synthesizing cycles remains mainly constant at a low level. The experiments were carried out at least 3 times for each condition (biological replications) in triplicates (technical replications).
Figure 4
Figure 4. Two-colour colocalisation microscopy.
Surface adsorbed 70S bioCANCy5 ribosomes (left) and formula image (right) images were acquired simultaneously at the same sample area. Colocalised ribosome (8 out of 49) and GFPem (8 out of 10) signals are marked (yellow circles). Scalebar: 5 μm.
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