UsingVibrio natriegensfor high-yield production of challenging expression targets and for protein deuteration

Abstract

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 bacteriumVibrio natriegensappeared as a novel and alternative host platform for production of proteins in high yields. Here, we used a commercial strain derived fromV. natriegens(VmaxTMX2) to produce soluble bacterial and fungal proteins in milligram scale, which we struggled to achieve inEscherichia coli. These proteins include the cholera toxin (CT) andN-acetyl glucosamine binding protein A (GbpA) fromVibrio cholerae, the heat-labile enterotoxin (LT) fromE. coliand the fungal nematotoxin CCTX2 fromCoprinopsis 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 inE. 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 inE. 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.

Producing recombinant proteins in Vibrio natriegens

“…3C). Similarly, increased yield allowed the consideration of isotopically labelled di cult-to-express proteins that would have been prohibitive to produce in E. coli, as was the case to produce 15 N RAF1(52-192) and deuterated KRAS4b (both reported here) and 13 C, 15 N, and deuterated RAF1(52-192) (data not shown).…”

“…Two-liter E. coli cultures were grown using the Dynamite medium protocol as described previously [17]. 15 N isotopic labeling of RAF1 RBD-CRD (52-192) Fifty milliliters of ZYM-20050-IO in a 250 mL ba ed ask was inoculated with an ice chip from a V. natriegens glycerol stock. This seed culture was incubated overnight at 30°C and shaken at 250 rpm (1" throw).…”

“…When puri cation yields drop below one milligram/liter, the cost of isotopically labeled proteins becomes prohibitive due to the larger fermentation volume (and thus reagent cost). As others have shown recently, V. natriegens can also be used to produce isotopically labelled proteins [6,15]. Thus, we explored producing isotopically labelled Hs.RAF1(52-192) in V. natriegens, hoping to reduce costs due to the higher yield.…”

“…Thus, we explored producing isotopically labelled Hs.RAF1(52-192) in V. natriegens, hoping to reduce costs due to the higher yield. This was investigated by comparing the yield from 15 N isotopic labeling fermentations between E. coli and V. natriegens (Table 1). While modest, the increase from 0.6 mg/L to 1.4 mg/L does merit the consideration of producing these more expensive reagents in the V. natriegens system.…”

“…The recent work cites fast growth rate, metabolic diversity, and the emerging development of tools for genetic manipulation as positive factors for the use of V. natriegens [15,16]. Fast growth rate was also what induced our lab to investigate the use of this system for recombinant protein production.…”