A Bioluminescent Agrobacterium tumefaciens for Imaging Bacterial Metabolic Activity in Planta.

Bioluminescence enables the monitoring of spatiotemporal dynamics and activity of bacterial populations in planta. We here describe a procedure to use AgroLux, a bioluminescent Agrobacterium tumefaciens, as a tool to study bacterial responses upon agroinfiltration. The first method details how to transform bioluminescent AgroLux to carry binary plasmids of interests.

AgroLux: bioluminescent Agrobacterium to improve molecular pharming and study plant immunity.

Agroinfiltration in Nicotiana benthamiana is widely used to transiently express heterologous proteins in plants. However, the state of Agrobacterium itself is not well studied in agroinfiltrated tissues, despite frequent studies of immunity genes conducted through agroinfiltration. Here, we generated a bioluminescent strain of Agrobacterium tumefaciens GV3101 to monitor the luminescence of Agrobacterium during agroinfiltration.

pH Gradient Mitigation in the Leaf Cell Secretory Pathway Attenuates the Defense Response of to Agroinfiltration.

Partial neutralization of the Golgi lumen pH by the ectopic expression of influenza virus M2 proton channel is useful to stabilize acid-labile recombinant proteins in plant cells, but the impact of pH gradient mitigation on host cellular functions has not been investigated. Here, we assessed the unintended effects of M2 expression on the leaf proteome of infiltrated with the bacterial gene vector . An isobaric tags for relative and absolute quantification quantitative proteomics procedure was followed to compare the leaf proteomes of plants agroinfiltrated with either an "empty" vector or an M2-encoding vector.

Proteases of Nicotiana benthamiana: an emerging battle for molecular farming.

Molecular farming increasingly uses the tobacco relative Nicotiana benthamiana for production of recombinant proteins through transient expression. Several proteins are produced efficiently with this expression platform, but yields for other proteins are often very low. These low yields are frequently due to endogenous proteases.

Activity-based proteomics reveals nine target proteases for the recombinant protein-stabilizing inhibitor SlCYS8 in Nicotiana benthamiana.

Co-expression of protease inhibitors like the tomato cystatin SlCYS8 is useful to increase recombinant protein production in plants, but key proteases involved in protein proteolysis are still unknown. Here, we performed activity-based protein profiling to identify proteases that are inhibited by SlCYS8 in agroinfiltrated Nicotiana benthamiana. We discovered that SlCYS8 selectively suppresses papain-like cysteine protease (PLCP) activity in both apoplastic fluids and total leaf extracts, while not affecting vacuolar-processing enzyme and serine hydrolase activity.

Recombinant protein susceptibility to proteolysis in the plant cell secretory pathway is pH-dependent.

Cellular engineering approaches have been proposed to mitigate unintended proteolysis in plant protein biofactories, involving the design of protease activity-depleted environments by gene silencing or in situ inactivation with accessory protease inhibitors. Here, we assessed the impact of influenza virus M2 proton channel on host protease activities and recombinant protein processing in the cell secretory pathway of Nicotiana benthamiana leaves. Transient co-expression assays with M2 and GFP variant pHluorin were first conducted to illustrate the potential of proton export from the Golgi lumen to promote recombinant protein yield.

Engineering Recombinant Virus-like Nanoparticles from Plants for Cellular Delivery.

Understanding capsid assembly following recombinant expression of viral structural proteins is critical to the design and modification of virus-like nanoparticles for biomedical and nanotechnology applications. Here, we use plant-based transient expression of the Bluetongue virus (BTV) structural proteins, VP3 and VP7, to obtain high yields of empty and green fluorescent protein (GFP)-encapsidating core-like particles (CLPs) from leaves. Single-particle cryo-electron microscopy of both types of particles revealed considerable differences in CLP structure compared to the crystal structure of infection-derived CLPs; in contrast, the two recombinant CLPs have an identical external structure.

An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves.

The overall quality of recombinant IgG antibodies in plants is dramatically compromised by host endogenous proteases. Different approaches have been developed to reduce the impact of endogenous proteolysis on IgGs, notably involving site-directed mutagenesis to eliminate protease-susceptible sites or the in situ mitigation of host protease activities to minimize antibody processing in the cell secretory pathway. We here characterized the degradation profile of H10, a human tumour-targeting monoclonal IgG, in leaves of Nicotiana benthamiana also expressing the human serine protease inhibitor α1-antichymotrypsin or the cysteine protease inhibitor tomato cystatin SlCYS8.

Production of a tumour-targeting antibody with a human-compatible glycosylation profile in N. benthamiana hairy root cultures.

Hairy root (HR) cultures derived from Agrobacterium rhizogenes transformation of plant tissues are an advantageous biotechnological manufacturing platform due to the accumulation of recombinant proteins in an otherwise largely protein free culture medium. In this context, HRs descending from transgenic Nicotiana tabacum plants were successfully used for the production of several functional mAbs with plant-type glycans. Here, we expressed the tumor-targeting monoclonal antibody mAb H10 in HRs obtained either by infecting a transgenic N.

A Chimeric Affinity Tag for Efficient Expression and Chromatographic Purification of Heterologous Proteins from Plants.

The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study, we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC) of plant-made proteins based on the protein-stabilizing fusion partner SlCYS8.

Companion Protease Inhibitors for the In Situ Protection of Recombinant Proteins in Plants.

We previously described a procedure for the use of plant protease inhibitors as "companion" accessory proteins to prevent unwanted proteolysis of clinically useful recombinant proteins in leaf crude protein extracts (Benchabane et al. Methods Mol Biol 483:265-273, 2009). Here we describe the use of these inhibitors for the protection of recombinant proteins in planta, before their extraction from leaf tissues.

Modulating secretory pathway pH by proton channel co-expression can increase recombinant protein stability in plants.

Eukaryotic expression systems are used for the production of complex secreted proteins. However, recombinant proteins face considerable biochemical challenges along the secretory pathway, including proteolysis and pH variation between organelles. As the use of synthetic biology matures into solutions for protein production, various host-cell engineering approaches are being developed to ameliorate host-cell factors that can limit recombinant protein quality and yield.