Insufficient accumulation levels of recombinant proteins in plants and the lack of efficient purification methods for recovering these valuable proteins have hindered the development of plant biotechnology applications. Hydrophobins are small and surface-active proteins derived from filamentous fungi that can be easily purified by a surfactant-based aqueous two-phase system. In this study, the hydrophobin HFBI sequence from Trichoderma reesei was fused to green fluorescent protein (GFP) and transiently expressed in Nicotiana benthamiana plants by Agrobacterium tumefaciens infiltration. The HFBI fusion significantly enhanced the accumulation of GFP, with the concentration of the fusion protein reaching 51% of total soluble protein, while also delaying necrosis of the infiltrated leaves. Furthermore, the endoplasmic reticulum-targeted GFP-HFBI fusion induced the formation of large novel protein bodies. A simple and scalable surfactant-based aqueous two-phase system was optimized to recover the HFBI fusion proteins from leaf extracts. The single-step phase separation was able to selectively recover up to 91% of the GFP-HFBI up to concentrations of 10 mg mL⁻¹. HFBI fusions increased the expression levels of plant-made recombinant proteins while also providing a simple means for their subsequent purification. This hydrophobin fusion technology, when combined with the speed and posttranslational modification capabilities of plants, enhances the value of transient plant-based expression systems.

Carbohydrate–protein interactions govern many crucial life processes involved in cell recognition events, but are often difficult to study because the interactions are weak, and multivalent exposure appears to be crucial for their biological function. We have used self-assembled monolayers (SAMs) of neoglycoconjugates as a model system to probe the specific interactions between the lectin wheat germ agglutinin (WGA) and monosaccharides by surface plasmon resonance (SPR) and atomic force microscopy (AFM) force measurements. SAMs presenting N-acetyl-d-glucosamine (GlcNAc) as a neoglycoconjugate were produced on gold surfaces, where the SAM formation was monitored using a quartz crystal microbalance (QCM) and shown to be a very rapid process. In the AFM force measurements WGA was covalently coupled to flexible polyethylene glycol (PEG) molecules at a probe surface using amine coupling. GlcNAc-specific binding events were detected with a WGA-modified probe on the GlcNAc-neoglycoconjugate SAM at bond rupture forces of 47 ± 15 pN. Additionally, less frequent GlcNAc-specific unbinding events were detected at higher forces (120 ± 20 pN) which are believed to originate from simultaneous detachment of multiple binding sites from the SAM surface. SPR measurements confirmed that WGA has higher affinity toward the immobilized GlcNAc-SAM than toward the soluble free monosaccharide. The binding constants obtained for soluble chitinoligosaccharides suggested up to three subsites within one carbohydrate-binding site of the WGA molecule and also provided further evidence of the multivalent binding character of the WGA dimer.

The production of edible microbial biomass in bioreactors is an attractive alternative to agriculture, which is a major consumer of fresh water, an emitter of greenhouse gases, and a cause of eutrophication. This study examines microbial food production by gas fermentation of two endotoxin-free Gram-positive H₂-oxidizing autotrophic bacterial strains, Nocardioides nitrophenolicus KGS-27 and Rhodococcus opacus DSM 43205. The supply of the gaseous substrates H₂ and O₂ by in situ water electrolysis was investigated as an alternative to providing them from an external source. N. nitrophenolicus KGS-27 produced ≤9.9 ± 2.0 mg of biomass L^–1 h^–1 and was not affected by in situ water electrolysis, which potentially produces growth-inhibiting reactive oxygen species. With R. opacus DSM 43205, in turn, electrolysis slowed growth considerably and resulted in a volumetric productivity of 2.6 ± 0.8 mg of biomass L^–1 h^–1, which was a quarter of what was obtained with the gases supplied externally. The macromolecular compositions of biomasses produced by the two cultivation modes using the two bacterial strains were rather similar containing approximately 33–37% protein, 28–56% carbohydrates, and 4–5% fatty acids of the cell dry weight. Relatively high protein concentrations and balanced amino acid profiles, comparable to those of casein and soy protein, were achieved with both strains.