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 Citations (Scopus)

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

Fingerprint

Cloning
Ribosomes
Organism Cloning
Complementary DNA
Display devices
Messenger RNA
Enzymes
Proteins
Gene Library
Substrates
G(M3) Ganglioside
G(M1) Ganglioside
Immobilized Enzymes
Transcription
Reverse Transcription
Purification
Amplification
Plasmids
Clone Cells
Binding Sites

Keywords

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

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Protein-ribosome-mRNA display : Affinity isolation of enzyme-ribosome-mRNA complexes and cDNA cloning in a single-tube reaction. / Bieberich, Erhard; Kapitonov, Dmitri; Tencomnao, Tewin; Yu, Robert K.

In: Analytical Biochemistry, Vol. 287, No. 2, 15.12.2000, p. 294-298.

Research output: Contribution to journalArticle

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