Quantitative screening of yeast surface-displayed polypeptide libraries by magnetic bead capture.
Magnetic bead capture is demonstrated here to be a feasible alternative for quantitative screening of favorable mutants from a cell-displayed polypeptide library. Flow cytometric sorting with fluorescent probes has been employed previously for high throughput screening for either novel binders or improved mutants. However, many laboratories do not have ready access to this technology as a result of the limited availability and high cost of cytometers, restricting the use of cell-displayed libraries. Using streptavidin-coated magnetic beads and biotinylated ligands, an alternative approach to cell-based library screening for improved mutants was developed. Magnetic bead capture probability of labeled cells is shown to be closely correlated with the surface ligand density. A single-pass enrichment ratio of 9400 +/- 1800-fold, at the expense of 85 +/- 6% binder losses, is achieved from screening a library that contains one antibody-displaying cell (binder) in 1.1 x 10(5) nondisplaying cells. Additionally, kinetic screening for an initial high affinity to low affinity (7.7-fold lower) mutant ratio of 1:95,000, the magnetic bead capture method attains a single-pass enrichment ratio of 600 +/- 200-fold with a 75 +/- 24% probability of loss for the higher affinity mutant. The observed high loss probabilities can be straightforwardly compensated for by library oversampling, given the inherently parallel nature of the screen. Overall, these results demonstrate that magnetic beads are capable of quantitatively screening for novel binders and improved mutants. The described methods are directly analogous to procedures in common use for phage display and should lower the barriers to entry for use of cell surface display libraries.
Yeung
Yik A
YA
Department of Chemical Engineering, and Division of Bioengineering and Environmental Health, Massachusetts Institute of Technology, Cambridge 02134, USA.
Wittrup
K Dane
KD
D000906:Antibodies / Q000235:genetics* / Q000378:metabolism; D000915:Antibody Affinity / Q000235:genetics; D005240:Feasibility Studies; D005434:Flow Cytometry / Q000379:methods*; D008024:Ligands; D008280:Magnetics; D008863:Microspheres; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D019151:Peptide Library; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D012680:Sensitivity and Specificity; D019809:Streptavidin
D000906:Antibodies; D008024:Ligands; D019151:Peptide Library; D019809:Streptavidin