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Efficiency of puromycin-based technologies mediated by release factors and a ribosome recycling factor. | LitMetric

Efficiency of puromycin-based technologies mediated by release factors and a ribosome recycling factor.

Protein Eng Des Sel

Division of Interactome Medical Sciences, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai Minato-ku, Tokyo 108-8639, Japan.

Published: August 2013

AI Article Synopsis

Article Abstract

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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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3711395PMC
http://dx.doi.org/10.1093/protein/gzt031DOI Listing

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