Microbead display by in vitro compartmentalisation: selection for binding using flow cytometry

FEBS Letters

Sepp, Armin¹  Griffiths, Andrew D¹  Tawfik, Dan S² 

[1] MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge CB2 2QH, UK;Centre for Protein Engineering, MRC Centre, Hills Road, Cambridge CB2 2QH, UK

关键词: In vitro compartmentalization;    Microbead;    Selection;    Binding;    Flow cytometry;    Directed evolution;    IVC;    in vitro compartmentalisation;    TSA;    tyramide signal amplification;    DHFR;    dihydrofolate reductase;   

DOI  :  10.1016/S0014-5793(02)03740-7

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

In vitro compartmentalisation in an emulsion was used to physically link proteins to the DNA that encodes them via microbeads. These microbeads can be selected for catalysis, or, as demonstrated here, for binding. Genes encoding a peptide containing an epitope (haemagglutinin) were enriched to near purity from a 10⁶-fold excess of genes encoding a different peptide by two rounds of selection using flow cytometry, indicating ∼1000-fold enrichment per round. Single beads can be isolated using flow sorting and the single gene on the bead amplified by polymerase chain reaction. Hence, the entire process can be performed completely in vitro.

【 授权许可】

Unknown