Key result
Using a reconstituted cell-free system without release factors and with ribosome recycling factor significantly increased the efficiency of C-terminal protein labelling and in vitro virus formation.
The study identifies optimal conditions for puromycin-based protein labelling and in vitro virus formation by manipulating release factors and ribosome recycling factors in a cell-free system.
Two puromycin-based techniques, in vitro virus (IVV) and C-terminal labelling of proteins, were developed based on the observation that puromycin binds the C-terminus of a protein. Puromycin technology is a useful tool for the detection of proteins and analysis of protein-protein interactions (PPIs); however, problems arise due to the existence of stop codons in the native mRNAs. Release factors (RFs) that enter the A-site of the ribosome at stop codons compete with puromycin. To overcome this issue, we have used a highly controllable reconstituted cell-free system for puromycin-based techniques, and observed efficient IVV formation and C-terminal labelling using templates possessing a stop codon. The optimal conditions of IVV formation using templates possessing a stop codon was RF (-), while that of C-terminal labelling was RF (-) and the ribosome recycling factor (RRF) (+). Thus, we have overcome the experimental limitations of conventional IVV. In addition, we discovered that RRF significantly increases the efficiency of C-terminal protein labelling, but not IVV formation.
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Ohashi et al. (2013) studied this question. Reconstituted cell-free system without release factors and with ribosome recycling factor vs. Conventional in vitro virus (IVV) system was evaluated on Efficiency of IVV formation and C-terminal labelling. Using a reconstituted cell-free system without release factors and with ribosome recycling factor significantly increased the efficiency of C-terminal protein labelling and in vitro virus formation.
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