An improved method for an efficient and easily accessible eukaryotic ribosome display technology.
Ribosome display is a powerful in vitro technology for the selection and directed evolution of proteins. However, this technology has so far been perceived as being technically challenging owing to comparatively difficult protocols and the absence of tailored commercial reagents, particularly when using prokaryotic cell-free expression systems. Eukaryotic ribosome display is potentially a more accessible alternative because of the availability of suitable commercial reagents, yet despite published protocols, this method has been less widely used. For eukaryotic ribosome display, a novel mechanism of mRNA recovery compared with that of the well-proven prokaryotic method has been proposed. We have examined the eukaryotic ribosome display process with the aims of investigating the proposed mechanism of sequence recovery and of identifying aspects of the protocol that may have lead to poor performance and therefore so far limited its use. We demonstrate that the proposed novel method is in fact mechanistically comparable to the prokaryotic method and we provide a step-by-step protocol for eukaryotic ribosome display that is 20-fold more efficient than current published methods. Our findings should increase the ease of operating ribosome display technology, making it more accessible to the scientific community.
Douthwaite
J A
JA
Cambridge Antibody Technology, Granta Park, Milstein Building, Cambridge CB1 6GH, UK. julie.douthwaite@cambridgeantibody.com
Groves
M A
MA
Dufner
P
P
Jermutus
L
L
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D066298:In Vitro Techniques; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D012331:RNA, Fungal / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D011817:Rabbits; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014158:Transcription, Genetic
D011506:Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D004247:DNA