Eukaryotic ribosome display system is widely used for selection of peptides/proteins, especially for the selection of functional single-chain antibody fragments. As a leader in ribosome display, Creative Biolabs offers coupled rabbit reticulocyte lysate system for ribosome display.
Ribosome display is a cell-free system for in vitro display. Ribosome serves as the connector to couple protein, corresponding mRNA to form stable protein-ribosome-mRNA (PRM) complexes. PRMs are exposed to immobilized target molecules to screening the right proteins. From those target-binding complexes, the mRNAs are isolated, reverse transcribed and amplified as DNA for further manipulation and protein expression. Ribosome display is an incomparable technique for displaying very large libraries.
Rabbit Reticulocyte Lysate System for Ribosome Display
The eukaryotic system is often used for the selection of antibody fragments using the coupled rabbit reticulocyte lysate. This system was initially termed ARM (Antibody-Ribosome-mRNA) display. The main difference between the E. coli S30 and rabbit reticulocyte ribosome display systems is in the DNA recovery step. E. coli S30 system needs chemicals (e.g. EDTA) for disrupting PRM complexes and releasing mRNA prior to RT-PCR, while the rabbit reticulocyte lysate system employs an in situ recovery procedure in which DNA was recovered directly on the ribosome complexes by amplification with a novel in situ RT-PCR procedure without dissociation of the ribosome complex.
Figure 1. ARM ribosome display for single-chain antibodies. T7: T7 promoter; PRM: protein-ribosome-mRNA; RT-PCR: coupled reverse transcription polymerase chain reaction; scAb: single chain antibody fragment; ARM: antibody-ribosome-mRNA. (He and Taussig 2002)
Successful in situ RT-PCR is achieved through the design of a novel internal primer which contains both a sequence for hybridizing to the upstream region of 3’ mRNA (to avoid the stalling ribosome) and a sequence identical to the 5’ region of mRNA. The novel internal primer can generate single-stranded cDNAs with a complementary flanking sequence at both 5’ and 3’ ends, which can be effectively amplified using a single primer.
Figure 2. In situ single primer RT-PCR recovery and the full length DNA regeneration. (He et al. 2012)
In situ RT-PCR not only simplifies the recovery process but also avoids material losses in the step of disrupting complexes for mRNA isolation. It has been used to analyze the binding specificity of PRM through detection of the attached mRNA. In situ RT-PCR would also facilitate automation of the ribosome display process.
The disruption method for releasing mRNA from rabbit reticulocyte ribosome complexes is different from that in prokaryotic ribosome display system (by EDTA). Generally, we disrupt rabbit reticulocyte complexes by heating above 70 °C.
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