Biopharmaceuticals, particularly monoclonal antibodies (mAbs), are a rapidly expanding class of therapeutics and have benefitted from high-throughput screening (HTS) strategies for protein engineering. Obtaining such complex biopharmaceuticals from plants might be advantageous because transient expression can yield grams of product within a week. For HTS, this system was scaled down as plant cell pack (PCP) technology to microtiter plate format and automated on a liquid-handling station, but is limited by manual cloning and protein purification. Therefore, we first generated a modular vector library comprising 15 pTRAc backbone constructs differing in regulatory gene expression and protein targeting elements, enabling rapid integration of any target gene via restriction-ligation cloning. Building on this, we developed an automated, seamless cloning workflow prior to transient expression and integrated a magnetic bead-based downstream purification process for single-chain fragment variable (scFv) mAbs. This automated workflow enables sequential cloning, expression, and purification of up to 375 protein variants simultaneously, each with 10 PCP replicates, within 9 days on a single platform. In contrast, manual processing allows only 15 variants with 10 replicates in the same period - a 25-fold increase in throughput, while costs decreased by 30%, from 79 € to 61 € per variant. Using a recombinant immunotoxin against acute myeloid leukemia based on the anti-CD64 H22 scFv as case study, a recovery of 63% and purity >95% were obtained, suitable for in vitro assays. Thus, integrating our cloning and purification workflows into the PCP platform establishes an automated pipeline that accelerates early biopharmaceutical development.
Monique Schulze, Patrick Opdensteinen, Sandor Albert et al.· New Biotechnology· 0 citations
Laccases are versatile oxidoreductases found in bacteria, fungi, and plants., which have a wide range of industrial applications, including the removal of pharmaceutical micropollutants from wastewater. Bacterial laccases are particularly attractive because of their high catalytic activity and stability over broad temperature and pH ranges. One example is the copper efflux oxidase (CueO) from
Escherichia coli
, which has been fused to the adhesion-promoting peptide Macaque Histatin (MacHis) because immobilized enzymes have proven especially effective for solid-phase wastewater treatment. However, the practical application of this enzyme is constrained by the relatively low production yields of 25–50 mg/L when expressed in recombinant
E. coli
or
Pichia pastoris
.
Here, we used transient expression in Nicotiana benthamiana to demonstrate that the engineered CueO-MacHis can be produced at substantially higher levels, reaching 250–320 (286 ± 35) mg/kg biomass in the prokaryotic-like environment of chloroplasts in 6-week-old plants, which are typically used for production purposes. Subsequent systematic optimization of the transient expression process based on statistical experimental designs increased the accumulation of recombinant CueO-MacHis approximately threefold to 846 ± 57 mg/kg biomass. Furthermore, we established a simplified and cost-effective purification strategy that leverages the enzyme’s temperature and pH stability to clarify extracts before anion exchange chromatography, achieving 76% recovery and 92% purity. Moreover, the plant-derived CueO-MacHis had a specific activity of 7.22 ± 1.55 U/mg, which is in the same range as that reported for other CueO enzymes.
Our approach paves the way for the large-scale production of CueO-MacHis as a technical enzyme for industrial applications.
Henrik Nausch, C. Bernau, Dirk Scheffler et al.· Frontiers in Plant Science· 0 citations