Robot Cookies – Plant Cell Packs as an Automated High-Throughput Screening Platform Based on Transient Expression

Abstract

The high-throughput screening of recombinant protein expression is advantageous during early process development because it allows the identification of optimal expression constructs and process conditions. Simple screening platforms based on microtiter plates are available for microbes and animal cells, but this was not possible for plants until the development of plant cell packs (PCPs), also known as "cookies," which provide a versatile and scalable screening tool for recombinant protein production. PCPs are prepared from plant cell suspension cultures by removing the medium and molding the biomass. PCPs can be cast into 96-well plates for highthroughput screening, but the manual handling effort currently limits the throughput to ∼500 samples per day. We have therefore integrated the PCP method with a fully automated laboratory liquid-handling station. The "robot cookies" can be prepared and infiltrated with Agrobacterium tumefaciens by centrifugation, minimizing operator handling and reducing the likelihood of errors during repeated runs, such as those required in a design of experiments approach. The accumulation of fluorescent protein in the cytosol, apoplast, endoplasmic reticulum or plastids is easily detected using an integrated plate reader, reducing the inter-experimental variation to <5%. We also developed a detergent-based chemical lysis method for protein extraction in a 96-well format, which was adapted for automated downstream processing using miniaturized columns allowing subsequent protein analysis. The new automated method reduces the costs of the platform to <0.5 € per PCP infiltration (a saving of >50%) and facilitates a five-fold increase in throughput to >2500 samples per day.

Expression of Biofilm-Degrading Enzymes in Plants and Automated High-Throughput Activity Screening Using Experimental Bacillus subtilis Biofilms

“…The coding sequence of the dispersin B homolog Lg2 from L. gummosus ( Gökçen, 2016 ) was modified to incorporate flanking N-terminal BspHI and C-terminal NotI sites before codon optimization for N. benthamiana and synthesis by GeneArt (Thermo Fisher Scientific, Darmstadt, Germany). Using the BspHI and NotI sites, the Lg2 gene was subcloned into 12 previously established pTRA vectors ( Gengenbach et al, 2020 ), originally derived from pPAM (GenBank AY027531), featuring all possible permutations ( Supplementary Table S1 ) of the CHS, omega, and TL 5’ untranslated regions (UTRs) combined with an LPH signal sequence targeting the secretory pathway, an rbcs signal sequence targeting the chloroplast, a SEKDEL ER retention signal, or none of the above to allow accumulation in the cytosol ( Buyel et al, 2013 ). Plasmids were propagated in E. coli DH5α and transferred to Agrobacterium tumefaciens ( Rhizobium radiobacter ) GV3101:pMP90RK by electroporation (2400 V, 25 μF, and 200 Ω) using 0.2 cm electroporation cuvettes (Bio-Rad Laboratories, Hercules, California, United States) as previously described ( Main et al, 1995 ).…”

“…Here, we optimized the upstream production of the dispersin B–like enzyme L. gummosus glyco 2 (Lg2) in plant cell packs (PCPs) ( Gengenbach et al, 2020 ) and transferred the optimal conditions to differentiated N. benthamiana plants for transient expression. We then developed an immobilized metal affinity chromatography (IMAC) protocol for the purification of Lg2.…”

The Emergency Response Capacity of Plant-Based Biopharmaceutical Manufacturing-What It Is and What It Could Be

“…The reproducibility of screening was improved in 2018 by the development of plant cell pack technology, in which plant cell suspension cultures deprived of medium are used to form a plant tissue surrogate that can be infiltrated with A. tumefaciens in a 96-well microtiter plate format to produce milligram quantities of protein in an automated, high-throughput manner. The costs (without analysis) can be as low as €0.50 (US$0.60) per 60-mg sample with a product accumulation of ~100 mg kg −1 and can typically result in a CV of <5% (Gengenbach et al, 2020). These costs include the fermenter-based upstream production of plant cells as well as all materials and labor.…”

Plant Molecular Farming as a Strategy Against COVID-19 – The Italian Perspective

“…The wide range of yields (1-220 µg/g fresh leaf mass, average 77.4 µg/g) shows that the feasibility of molecular farming for antigen manufacturing is exquisitely sensitive to the intrinsic nature of the product candidate (and also the quantification method). It is not yet possible to accurately predict yields based on a given candidate protein sequence, and empirical evaluation is therefore necessary, including the testing of multiple expression strategies -which is also facilitated by the scalability of transient expression systems (Gengenbach et al, 2020). Most immunological diagnostic tests proposed for the detection of SARS-CoV-2 antibodies are based on the fulllength viral spike (S) glycoprotein, the shorter external S1 segment, its receptor binding domain (RBD), or the N protein (Freeman et al, 2020;Klumpp-Thomas et al, 2020;Rosendal et al, 2020).…”

“…The wide range of yields (1–220 μg/g fresh leaf mass, average 77.4 μg/g) shows that the feasibility of molecular farming for antigen manufacturing is exquisitely sensitive to the intrinsic nature of the product candidate (and also the quantification method). It is not yet possible to accurately predict yields based on a given candidate protein sequence, and empirical evaluation is therefore necessary, including the testing of multiple expression strategies – which is also facilitated by the scalability of transient expression systems ( Gengenbach et al, 2020 ).…”