Protein–Ribosome–mRNA Display: Affinity Isolation of Enzyme–Ribosome–mRNA Complexes and cDNA Cloning in a Single-Tube Reaction
✍ Scribed by Erhard Bieberich; Dmitri Kapitonov; Tewin Tencomnao; Robert K. Yu
- Publisher
- Elsevier Science
- Year
- 2000
- Tongue
- English
- Weight
- 82 KB
- Volume
- 287
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
An enzyme-ribosome-mRNA complex was specifically purified by binding to the immobilized enzyme substrate and the cDNA was cloned in a single-tube reaction by one-step reverse transcription-PCR. The ganglioside GM3, used by sialyltransferase II (ST-II) as a substrate, was coated on a 96-well microtiter plate and ST-II was in vitro transcribed and translated from a cDNA library. The isolation of an enzyme-specific protein-ribosome (PRIME) complex was achieved with as little as 0.1 ng ST-II-specific cDNA in 5 g of a total plasmid preparation or with the cDNA prepared from sublibraries previously inoculated at a density of 2000 clones/culture well. The affinity purification of the PRIME complex was highly specific for GM3 and did not result in cDNA amplification when a different ganglioside (GM1) was used for coating of the microtiter plate. The amplified cDNA was used for cloning or a second round of ribosome display, providing a fast analysis of enzyme affinity to multiple substrates. PRIME display can be used for host-free cDNA cloning from mRNA or cDNA libraries and for binding site mapping of the in vitro translated protein. The use of a single-tube reaction in ligand-coated microtiter plates indicates the versatility of PRIME display for cDNA cloning by automated procedures.