Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation.

The equilibrium binding affinity of receptor-ligand or antibody-antigen pairs may be modulated by protonation of histidine side-chains, and such pH-dependent mechanisms play important roles in biological systems, affecting molecular uptake and trafficking. Here, we aimed to manipulate cellular transport of single-chain antibodies (scFvs) against the transferrin receptor (TfR) by engineering pH-dependent antigen binding. An anti-TfR scFv was subjected to histidine saturation mutagenesis of a single CDR.

Yeast Display-Based Antibody Affinity Maturation Using Detergent-Solubilized Cell Lysates.

It is often desired to identify or engineer antibodies that target membrane proteins (MPs). However, due to their inherent insolubility in aqueous solutions, MPs are often incompatible with in vitro antibody discovery and optimization platforms. Recently, we adapted yeast display technology to accommodate detergent-solubilized cell lysates as sources of MP antigens.

An evolved Mxe GyrA intein for enhanced production of fusion proteins.

Expressing antibodies as fusions to the non-self-cleaving Mxe GyrA intein enables site-specific, carboxy-terminal chemical modification of the antibodies by expressed protein ligation (EPL). Bacterial antibody-intein fusion protein expression platforms typically yield insoluble inclusion bodies that require refolding to obtain active antibody-intein fusion proteins. Previously, we demonstrated that it was possible to employ yeast surface display to express properly folded single-chain antibody (scFv)-intein fusions, therefore permitting the direct small-scale chemical functionalization of scFvs.

Antibody affinity maturation using yeast display with detergent-solubilized membrane proteins as antigen sources.

Antigen preparations in the form of detergent-solubilized cell lysates could, in principle, render membrane proteins (MPs) compatible with in vitro antibody engineering technologies. To this end, detergent-solubilized cell lysates were coupled with the yeast surface display platform to affinity mature an anti-transferrin receptor (TfR) single-chain antibody (scFv). Lysates were generated from TfR-expressing HEK293 cells by solubilization with detergent-containing buffer after undergoing plasma membrane-restricted biotinylation.

Cells and cell lysates: a direct approach for engineering antibodies against membrane proteins using yeast surface display.

Membrane proteins (MPs) are often desirable targets for antibody engineering. However, the majority of antibody engineering platforms depend implicitly on aqueous solubility of the target antigen which is often problematic for MPs. Recombinant, soluble forms of MPs have been successfully employed as antigen sources for antibody engineering, but heterologous expression and purification of soluble MP fragments remains a challenging and time-consuming process.