Protein-ribosome-mRNA display: Affinity isolation of enzyme-ribosome-mRNA complexes and cDNA cloning in a single-tube reaction

Erhard Bieberich, Dmitri Kapitonov, Tewin Tencomnao, Robert K. Yu

Research output: Contribution to journalArticle

24 Scopus citations

Abstract

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 μg 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.

Original languageEnglish (US)
Pages (from-to)294-298
Number of pages5
JournalAnalytical Biochemistry
Volume287
Issue number2
DOIs
StatePublished - Dec 15 2000

Keywords

  • CDNA cloning
  • Functional genomics
  • Ganglioside
  • Glycosyltransferase
  • Ribosome display

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Protein-ribosome-mRNA display: Affinity isolation of enzyme-ribosome-mRNA complexes and cDNA cloning in a single-tube reaction'. Together they form a unique fingerprint.

  • Cite this