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

Autor:	Mojica N; Department of Chemistry, University of Oslo, Blindern, Norway., Kersten F; Department of Chemistry, University of Oslo, Blindern, Norway.; Centre for Molecular Medicine Norway, University of Oslo, Blindern, Norway., Montserrat-Canals M; Department of Chemistry, University of Oslo, Blindern, Norway.; Centre for Molecular Medicine Norway, University of Oslo, Blindern, Norway., Huhn GR 3rd; Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida, U.S.A., Tislevoll AM; Department of Chemistry, University of Oslo, Blindern, Norway., Cordara G; Department of Chemistry, University of Oslo, Blindern, Norway., Teter K; Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida, U.S.A., Krengel U; Department of Chemistry, University of Oslo, Blindern, Norway.
Jazyk:	angličtina
Zdroj:	BioRxiv : the preprint server for biology [bioRxiv] 2023 Nov 03. Date of Electronic Publication: 2023 Nov 03.
DOI:	10.1101/2023.11.03.565449
Abstrakt:	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. Competing Interests: CONFLICT OF INTEREST The authors declare that they have no conflicts of interest with the contents of this article.
Databáze:	MEDLINE
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