Protein-ribosome-mRNA display: affinity isolation of enzyme-ribosome-mRNA complexes and cDNA cloning in a single-tube reaction.
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 microg 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.
Bieberich
E
E
Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th Street CB-2803, Augusta, Georgia 30912, USA.
Kapitonov
D
D
Tencomnao
T
T
Yu
R K
RK
D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000378:metabolism*; D012799:Sialyltransferases / Q000302:isolation & purification / Q000378:metabolism*
D018076:DNA, Complementary; D012333:RNA, Messenger; D012799:Sialyltransferases; C043662:alpha-N-acetylneuraminate alpha-2,8-sialyltransferase