Microbeads display of proteins using emulsion PCR and cell‐free protein synthesis
2008; American Chemical Society; Volume: 24; Issue: 5 Linguagem: Inglês
10.1002/btpr.43
ISSN8756-7938
AutoresRui Gan, Yumiko Yamanaka, Takaaki Kojima, Hideo Nakano,
Tópico(s)Bacteriophages and microbial interactions
ResumoAbstract We developed a method for coupling protein to its coding DNA on magnetic microbeads using emulsion PCR and cell‐free protein synthesis in emulsion. A PCR mixture containing streptavidin‐coated microbeads was compartmentalized by water‐in‐oil (w/o) emulsion with estimated 0.5 template molecules per droplet. The template molecules were amplified and immobilized on beads via bead‐linked reverse primers and biotinylated forward primers. After amplification, the templates were sequentially labeled with streptavidin and biotinylated anti‐glutathione S‐transferase (GST) antibody. The pool of beads was then subjected to cell‐free protein synthesis compartmentalized in another w/o emulsion, in which templates were coupled to their coding proteins. We mixed two types of DNA templates of Histidine6 tag (His6)‐fused and FLAG tag‐fused GST in a ratio of 1:1,000 (His6: FLAG) for use as a model DNA library. After incubation with fluorescein isothiocyanate (FITC)‐labeled anti‐His6 (C‐term) antibody, the beads with the His6 gene were enriched 917‐fold in a single‐round screening by using flow cytometry. A library with a theoretical diversity of 10 6 was constructed by randomizing the middle four residues of the His6 tag. After a two‐round screening, the randomized sequences were substantially converged to peptide‐encoding sequences recognized by the anti‐His6 antibody.
Referência(s)