ALiCE^®: A versatile, high yielding and scalable eukaryotic cell-free protein synthesis (CFPS) system

Abstract

Eukaryotic cell-free protein synthesis (CFPS) systems have the potential to simplify and speed up the expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and the inability to scale such systems have so far prevented their widespread adoption in protein research and manufacturing. Here, we present a detailed demonstration 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 under 48 hours, complete with native disulfide bonds and N-glycosylation. An optimised version of the technology is commercialised as 'ALiCE', engineered for high yields of up to 3 mg/mL. Recent advances in the scaling of BYL production methodologies have allowed scaling of the CFPS reaction. We show simple, linear scale-up of batch mode reporter proten expression from a 100 uL microtiter plate format to 10 mL and 100 mL volumes in standard Erlenmeyer flasks, culminating in preliminary data from 1 L reactions in a CELL-tainer CT20 rocking motion bioreactor. As such, these works represent the first published example of a eukaryotic CFPS reaction scaled past the 10 mL level by several orders of magnitude. We show the ability of BYL to produce the simple reporter protein eYFP and large, multimeric virus-like particles directly in the cytosolic fraction. Complex proteins are processed using the native microsomes of BYL and functional expression of multiple classes of complex, difficult-to-express proteins is demonstrated, specifically: a dimeric, glycoprotein enzyme, glucose oxidase; the monoclonal antibody adalimumab; the SARS-Cov-2 receptor-binding domain; human epidermal growth factor; and a G protein-coupled receptor membrane protein, cannabinoid receptor type 2. Functional binding and activity are shown using a combination of surface plasmon resonance techniques, a serology-based ELISA method and a G protein activation assay. Finally, in-depth post-translational modification (PTM) characterisation of purified proteins through disulfide bond and N-glycan analysis is also revealed - previously difficult in the eukaryotic CFPS space due to limitations in reaction volumes and yields. Taken together, BYL provides a real opportunity for screening of complex proteins at the microscale with subsequent amplification to manufacturing-ready levels using off-the-shelf protocols. This end-to-end platform suggests the potential to significantly reduce cost and the time-to-market for high value proteins and biologics.

IRES-mediated Pichia pastoris cell-free protein synthesis

“…3 D) that the rate of sfGFP synthesis was highest in the first 1 h and increased almost linearly. The rate of protein synthesis decreased from 1 to 6 h, and there was no significant increase in fluorescence after 6 h. Therefore, the optimal time to achieve high yield with minimum time consumption was determined to be 6 h. Previous eukaryotic CFPS studies have shown that the reaction time for tobacco CFPS takes 48 h and 12 h for CHO CFPS (Das Gupta et al 2022 ; Heide et al 2021 ). Thus, the P. pastoris system has a shorter reaction time compared to other eukaryotic CFPS systems.…”

Amino acid balancing for the prediction and evaluation of protein concentrations in cell‐free protein synthesis systems

“…Also, a eukaryotic system based on Nicotiana tabacum reaches protein concentrations of up to 3 mg mL À1 and is commercialized as ALiCE ® (Almost Living Cell-free Expression). 46 The peculiarity of this system is that it contains native, actively translocating microsomal vesicles derived from the endoplasmic reticulum and the Golgi and shows good capabilities of performing post-translational modifications. For this system, however, no F I G U R E 3 Achieved protein concentrations (dark gray) and fractional yield (light gray) of CFPS systems starting from (a) different organisms or (b) same organisms from different studies.…”

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].…”