Using Vibrio natriegens for High-Yield Production of Challenging Expression Targets and for Protein Perdeuteration.

Production of soluble proteins is essential for structure/function studies; however, this usually requires milligram amounts of protein, which can be difficult to obtain with traditional expression systems. Recently, the Gram-negative bacterium Vibrio natriegens emerged as a novel and alternative host platform for production of proteins in high yields. Here, we used a commercial strain derived from V. natriegens (Vmax X2) to produce soluble bacterial and fungal proteins in milligram scale, which we struggled to achieve in Escherichia coli. These proteins include the cholera toxin (CT) and N-acetyl glucosamine-binding protein A (GbpA) from Vibrio cholerae, the heat-labile enterotoxin (LT) from E. coli and the fungal nematotoxin CCTX2 from Coprinopsis cinerea. CT, GbpA, and LT are secreted by the Type II secretion system in their natural hosts. When these three proteins were produced in Vmax, they were also secreted and could be recovered from the growth media. This simplified the downstream purification procedure and resulted in considerably higher protein yields compared to production in E. coli (6- to 26-fold increase). We also tested Vmax for protein perdeuteration using deuterated minimal media with deuterium oxide as solvent and achieved a 3-fold increase in yield compared to the equivalent protocol in E. coli. This is good news, since isotopic labeling is expensive and often ineffective but represents a necessary prerequisite for some structural biology techniques. Thus, Vmax represents a promising host for production of challenging expression targets and for protein perdeuteration in amounts suitable for structural biology studies.

Using Vibrio natriegens for high-yield production of challenging expression targets and for protein deuteration.

Production of soluble proteins is essential for structure/function studies, however, this usually requires milligram amounts of protein, which can be difficult to obtain with traditional expression systems. Recently, the Gram-negative bacterium Vibrio natriegens appeared as a novel and alternative host platform for production of proteins in high yields. Here, we used a commercial strain derived from V. natriegens (Vmax™ X2) to produce soluble bacterial and fungal proteins in milligram scale, which we struggled to achieve in Escherichia coli. These proteins include the cholera toxin (CT) and N-acetyl glucosamine binding protein A (GbpA) from Vibrio cholerae, the heat-labile enterotoxin (LT) from E. coli and the fungal nematotoxin CCTX2 from Coprinopsis cinerea. CT, GbpA and LT are secreted by the Type II secretion system in their natural hosts. When these three proteins were produced in Vmax, they were also secreted, and could be recovered from the growth media. This simplified the downstream purification procedure and resulted in considerably higher protein yields compared to production in E. coli (6- to 26-fold increase). We also tested Vmax for protein deuteration using deuterated minimal media with deuterium oxide as solvent, and achieved a 3-fold increase in yield compared to the equivalent protocol in E. coli. This is good news since isotopic labeling is expensive and often ineffective, but represents a necessary prerequisite for some structural techniques. Thus, Vmax represents a promising host for production of challenging expression targets and for protein deuteration in amounts suitable for structural biology studies.

Perdeuterated GbpA Enables Neutron Scattering Experiments of a Lytic Polysaccharide Monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) are surface-active redox enzymes that catalyze the degradation of recalcitrant polysaccharides, making them important tools for energy production from renewable sources. In addition, LPMOs are important virulence factors for fungi, bacteria, and viruses. However, many knowledge gaps still exist regarding their catalytic mechanism and interaction with their insoluble, crystalline substrates. Moreover, conventional structural biology techniques, such as X-ray crystallography, usually do not reveal the protonation state of catalytically important residues. In contrast, neutron crystallography is highly suited to obtain this information, albeit with significant sample volume requirements and challenges associated with hydrogen's large incoherent scattering signal. We set out to demonstrate the feasibility of neutron-based techniques for LPMOs using N-acetylglucosamine-binding protein A (GbpA) from Vibrio cholerae as a target. GbpA is a multifunctional protein that is secreted by the bacteria to colonize and degrade chitin. We developed an efficient deuteration protocol, which yields >10 mg of pure 97% deuterated protein per liter expression media, which was scaled up further at international facilities. The deuterated protein retains its catalytic activity and structure, as demonstrated by small-angle X-ray and neutron scattering studies of full-length GbpA and X-ray crystal structures of its LPMO domain (to 1.1 Å resolution), setting the stage for neutron scattering experiments with its substrate chitin.

Structure prediction of honey bee vitellogenin: a multi-domain protein important for insect immunity.

Vitellogenin (Vg) has been implicated as a central protein in the immunity of egg-laying animals. Studies on a diverse set of species suggest that Vg supports health and longevity through binding to pathogens. Specific studies of honey bees (Apis mellifera) further indicate that the vitellogenin (vg) gene undergoes selection driven by local pathogen pressures. Determining the complete 3D structure of full-length Vg (flVg) protein will provide insights regarding the structure-function relationships underlying allelic variation. Honey bee Vg has been described in terms of function, and two subdomains have been structurally described, while information about the other domains is lacking. Here, we present a structure prediction, restrained by experimental data, of flVg from honey bees. To achieve this, we performed homology modeling and used AlphaFold before using a negative-stain electron microscopy map to restrict, orient, and validate our 3D model. Our approach identified a highly conserved Ca2+ -ion-binding site in a von Willebrand factor domain that might be central to Vg function. Thereafter, we used rigid-body fitting to predict the relative position of high-resolution domains in a flVg model. This mapping represents the first experimentally validated full-length protein model of a Vg protein and is thus relevant for understanding Vg in numerous species. Our results are also specifically relevant to honey bee health, which is a topic of global concern due to rapidly declining pollinator numbers.