Rapid Affinity Characterization of Cell-Free Expressed Nanobodies Directly in Lysate with Biolayer Interferometry via...

There have been a few recent works showing the advantages of combining cell-free protein expression (CFE) with biolayer interferometry (BLI) for the rapid kinetic characterization of novel capture proteins, such as nanobodies, to build out sequence-to-function data sets. Such data can inform iterative, machine-learning-guided models to optimize binding to a target antigen. In this work, we extend these approaches by presenting a new CFE-BLI workflow that flips the BLI binding orientation so that the target protein is immobilized on the biosensor surface and the CFE-expressed nanobody is measured directly in the lysate. This expands the applicability of CFE-BLI screening to encompass a larger set of nanobody-antigen pairs, such as multimeric target proteins. Our workflow utilizes a fluorogenic HaloTag fusion protein that enables precise protein quantification while also amplifying the BLI signal of smaller-molecular-weight binders like nanobodies. We test our workflow here using two previously characterized nanobodies (I3 and 2Rs15d) and show that our CFE-BLI screening method can calculate equilibrium dissociation constants (K_D) similar to those previously reported. We further combine our workflow with a modern combinatorial mutagenesis workflow to screen 12 previously uncharacterized I3 nanobody mutants for improved affinity, demonstrating its potential use in rapid, high-iteration active-learning-guided protein engineering campaigns, especially on multimeric targets that may be presented in complex backgrounds.