Highly avid magnetic bead capture: An efficient selection method for de novo protein engineering utilizing yeast surface display

Abstract

Protein engineering relies on the selective capture of members of a protein library with desired properties. Yeast surface display technology routinely enables as much as million-fold improvements in binding affinity by alternating rounds of diversification and flow cytometry-based selection. However, flow cytometry is not well suited for isolating de novo binding clones from naïve libraries due to limitations in the size of the population that can be analyzed, the minimum binding affinity of clones that can be reliably captured, the amount of target antigen required, and the likelihood of capturing artifactual binders to the reagents. Here, we demonstrate a method for capturing rare clones that maintains the advantages of yeast as the expression host, while avoiding the disadvantages of FACS in isolating de novo binders from naïve libraries. The multivalency of yeast surface display is intentionally coupled with multivalent target presentation on magnetic beads-allowing isolation of extremely weak binders from billions of non-binding clones, and requiring far less target antigen for each selection, while minimizing the likelihood of isolating undesirable alternative solutions to the selective pressure. Multivalent surface selection allows 30,000-fold enrichment and almost quantitative capture of micromolar binders in a single pass using less than one microgram of target antigen. We further validate the robust nature of this selection method by isolation of de novo binders against lysozyme as well as its utility in negative selections by isolating binders to streptavidin-biotin that do not cross-react to streptavidin alone.

Highly Stable Binding Proteins Derived from the Hyperthermophilic Sso7d Scaffold

“…This magnetic selection procedure allows capture of even low-affinity binders owing to the avidity effect arising from the interaction between multiple copies of the Sso7d mutant protein on the yeast cell surface and multiple target molecules immobilized on the magnetic beads. 27 Before isolation of the binders, negative selection steps using magnetic beads or beads coated with non-target proteins were used. This step enables the rejection of yeast cells displaying Sso7d mutants that bind the beads and non-target proteins from further analysis.…”

“…27 Yeast cells grown in SDCAA were pelleted and suspended at 1 × 10 7 cells/ml (A 600 1) in SGCAA(20 g/l galactose, 5 g/l Casamino acids, 6.7 g/l yeast nitrogen base, 5.40 g/l Na 2 HPO 4 , 7.45 g/l NaH 2 PO 4 ). Cells were incubated at 20°C and 250 rpm for 20-24 h to induce expression of yeast cell surface protein fusions.…”

Stability and CDR Composition Biases Enrich Binder Functionality Landscapes

“…The libraries were pooled for comparison and tested for their ability to generate binders to seven targets: human A33, mouse A33, epidermal growth factor receptor (EGFR), Fcγ receptors IIA and IIIA (FcγRIIA and FcγRIIIA), mouse immunoglobulin G (mIgG), and human serum albumin (HSA). The naïve library was sorted by magnetic bead selections, 27 and lead clones were diversified by error-prone PCR on the full Fn3 gene and shuffling of mutagenized Fn3 loops. 25 Multiple rounds of diversification and selection, by magnetic beads and ultimately flow cytometry (Supplementary Fig.…”

“…Binders to streptavidin-coated magnetic Dynabeads (Invitrogen) were removed. 35 Biotinylated protein was loaded on streptavidin-coated magnetic Dynabeads and incubated with the remaining yeast. The beads were washed with phosphate-buffered saline with bovine serum albumin (PBSA), and the beads with attached cells were grown for further selection.…”

Antibody-Mediated Neutralization of Perfringolysin O for Intracellular Protein Delivery

“…A pooled combination of the YS, G2, and G4 Fn3 libraries previously developed 29 were first screened with biotinylated PFO captured on magnetic beads, 30 followed by fluorescence-activated cell sorting (FACS). Random mutagenesis was performed after every two or three selections to maintain a high library diversity.…”