Construction and Screening of Antigen Targeted Immune Yeast Surface Display Antibody Libraries

Abstract

These protocols describe a yeast surface display-based process for the rapid selection of antibodies from immunized mice, eliminating the need for creating and screening hybridoma fusions. A yeast surface display library of single-chain antibody fragments (scFvs) is created from antigen-binding B cells from the splenocytes of immunized mice. The antigen targeted library is then screened for antigen specific scFv by magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Library construction and screening can be accomplished in as little as 2 weeks, resulting in a panel of scFvs specific for the target antigen.

Affinity Maturation to Improve Human Monoclonal Antibody Neutralization Potency and Breadth against Hepatitis C Virus

“…Two rounds of enrichment were carried out on magnetic bead columns using an equilibrium method with the secreted E2 as the medium base (43). For the first round, we used secreted 1b34 E2 in a 1:5 dilution, with biotin-CBH-4G (an hmAb to a non-neutralizing epitope on HCV E2) (44) as the detection antibody and streptavidin-magnetic microbeads to capture the E2-bound yeast.…”

Engineering New Protein–Protein Interactions on the β‐Propeller Fold by Yeast Cell Surface Display

“…We cloned the KR of Keap1 into the yeast vector pCTCON2 to express KR as a fusion to agglutinin protein Aga2p that is attached to the yeast cell wall through disulfide bonds to Aga1p (Figure 2A). [20–21] To measure the display level of KR on the cell surface, we incubated the cells with a mouse anti-HA antibody and a chicken anti-Myc antibody that would bind to the cognate peptide tags fused to the N- and C-termini of KR, respectively. We then incubated the cells with a mixture of a goat anti-mouse antibody conjugated with Alexa Fluor 647 and a goat anti-chicken antibody conjugated with Alexa Fluor 488 to label the tags with distinct fluorophores (Figure 2A).…”

Engineering the Substrate Specificity of the DhbE Adenylation Domain by Yeast Cell Surface Display

“…Yeast cell surface display has been extensively used to engineer the binding specificity of antibodies (Chao et al, 2006; Miller et al, 2008). The yeast vector pCTCON2 expresses the antibody library as a fusion to the yeast agglutinin protein Aga2p that is attached through disulfide bonds to Aga1p protein as part of the yeast cell wall (Figure 1B).…”

“…The yeast selection followed the protocol developed by Wittrup and Miller with some modifications (Chao et al, 2006; Miller et al, 2008). In the first round of selection we incubated the yeast cell library with 3 μM 9 .…”