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

Abstract

Eukaryotic 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.

Establishing a High-Yield Chloroplast Cell-Free System for Prototyping Genetic Parts

“…Our chloroplast-based system is unique from the previously published tobacco BY-2 cell lysate system, , which is a highly productive expression system capable of combined transcription, translation, and protein modification. Specifically, the BY-2 lysate system is a eukaryotic chromosomal system and thus is different.…”

A Cost-Effective Pichia pastoris Cell-Free System Driven by Glycolytic Intermediates Enables the Production of Complex Eukaryotic Proteins

“…Although E.coli cell-free protein synthesis is widely used, the prokaryotic cell lysate offers limited post-translational modifications and thus fewer issues in the production of complex proteins [19]. On the other hand, post-translational modifications and optimal folding conditions are possible in eukaryotic cell-free protein production systems based on mammalian, insect, and tobacco cells [20][21][22]. Moreover, it was shown that membrane proteins can be translocated into vesicles derived from the endoplasmic reticulum (ER), termed microsomes, to facilitate the production of active ion channels in CHO and insect cell-free systems [23].…”

Cell-free expression and SMA copolymer encapsulation of a functional receptor tyrosine kinase disease variant, FGFR3-TACC3

“…Eukaryotic lysates can include machinery capable of PTMs and yields have been increased thanks to the adoption of T7-polymerase systems to allow coupled transcription-translation within the same system [6]. Notably, a CFPE system based on Nicotiana tabacum BY-2 lysate is capable of PTM incorporation, high specific yields, and can be effectively scaled between the microlitre and litre ranges [7,8]. Such systems allow the expression of eYFP at up to 3 mg/ml lysate used over a 48-hour synthesis [8].…”