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 high-throughput 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.
Keywords: Agrobacterium tumefaciens; automated transient expression; design of experiments; plant molecular farming; rapid protein synthesis; small-scale production.
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