Scaling eukaryotic cell‐free protein synthesis achieved with the versatile and high‐yielding tobacco BY‐2 cell lysate

Abstract

AbstractEukaryotic cell‐free protein synthesis (CFPS) can accelerate expression and high‐throughput analysis of complex proteins with functionally relevant post‐translational modifications (PTMs). However, low yields and difficulties scaling such systems have prevented their widespread adoption in protein research and manufacturing.Here, we provide detailed demonstrations for the capabilities of a CFPS system derived from Nicotiana tabacum BY‐2 cell culture (BY‐2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in 48 h, complete with native disulfide bonds and N‐glycosylation. An optimized version of the technology is commercialized as ALiCE® and advances in scaling of BYL production methodologies now allow scaling of eukaryotic CFPS reactions. We show linear, lossless scale‐up of batch mode protein expression from 100 µL microtiter plates to 10 and 100 mL volumes in Erlenmeyer flasks, culminating in preliminary data from a litre‐scale reaction in a rocking‐type bioreactor. Together, scaling across a 20,000x range is achieved without impacting product yields.Production of multimeric virus‐like particles from the BYL cytosolic fraction were then shown, followed by functional expression of multiple classes of complex, difficult‐to‐express proteins using the native microsomes of the BYL CFPS. Specifically: a dimeric enzyme; a monoclonal antibody; the SARS‐CoV‐2 receptor‐binding domain; a human growth factor; and a G protein‐coupled receptor membrane protein. Functional binding and activity are demonstrated, together with in‐depth PTM characterization of purified proteins through disulfide bond and N‐glycan analysis.Taken together, BYL is a promising end‐to‐end R&D to manufacturing platform with the potential to significantly reduce the time‐to‐market for high value proteins and biologics.