Microbeads display of proteins using emulsion PCR and cell-free protein synthesis.
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.
Gan
Rui
R
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.
Yamanaka
Yumiko
Y
Kojima
Takaaki
T
Nakano
Hideo
H
D002474:Cell-Free System; D004247:DNA / Q000737:chemistry; D004655:Emulsions; D015723:Gene Library; D008280:Magnetics; D008863:Microspheres; D016133:Polymerase Chain Reaction / Q000379:methods*; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry; D013499:Surface Properties
D004655:Emulsions; D011506:Proteins; D004247:DNA; D019809:Streptavidin