High yield expression and purification of isotopically labelled human endothelin-1 for use in NMR studies

Mac, Thien-Thi; Beyermann, Michael; Pires, Juliana Rico; Schmieder, Peter; Oschkinat, Hartmut

doi:10.1016/j.pep.2006.01.022

Cited by 8 publications

(2 citation statements)

References 18 publications

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“…Bogomolovas et al reported the production of active viscotoxin A3 containing three disulfide bonds using thioredoxin as a fusion partner [13]. Similarly, Mac et al reported production of Endothelin-1 containing two disulfides using thioredoxin [64], while Tedford et al used a GST-fusion for the production of an insecticidal spider toxin containing three disulfide bonds [63]. Our present work shows that many other DRPs can be produced and purified to homogeneity at the milligram scale using our protocols, spanning a diversity of folds (α/β, ICK, Kazal type, Kunitz).…”

Section: Discussionmentioning

confidence: 99%

High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli

et al. 2013

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BackgroundDisulfide-rich proteins or DRPs are versatile bioactive compounds that encompass a wide variety of pharmacological, therapeutic, and/or biotechnological applications. Still, the production of DRPs in sufficient quantities is a major bottleneck for their complete structural or functional characterization. Recombinant expression of such small proteins containing multiple disulfide bonds in the bacteria E. coli is considered difficult and general methods and protocols, particularly on a high throughput scale, are limited.ResultsHere we report a high throughput screening approach that allowed the systematic investigation of the solubilizing and folding influence of twelve cytoplasmic partners on 28 DRPs in the strains BL21 (DE3) pLysS, Origami B (DE3) pLysS and SHuffle® T7 Express lysY (1008 conditions). The screening identified the conditions leading to the successful soluble expression of the 28 DRPs selected for the study. Amongst 336 conditions tested per bacterial strain, soluble expression was detected in 196 conditions using the strain BL21 (DE3) pLysS, whereas only 44 and 50 conditions for soluble expression were identified for the strains Origami B (DE3) pLysS and SHuffle® T7 Express lysY respectively. To assess the redox states of the DRPs, the solubility screen was coupled with mass spectrometry (MS) to determine the exact masses of the produced DRPs or fusion proteins. To validate the results obtained at analytical scale, several examples of proteins expressed and purified to a larger scale are presented along with their MS and functional characterization.ConclusionsOur results show that the production of soluble and functional DRPs with cytoplasmic partners is possible in E. coli. In spite of its reducing cytoplasm, BL21 (DE3) pLysS is more efficient than the Origami B (DE3) pLysS and SHuffle® T7 Express lysY trxB-/gor- strains for the production of DRPs in fusion with solubilizing partners. However, our data suggest that oxidation of the proteins occurs ex vivo. Our protocols allow the production of a large diversity of DRPs using DsbC as a fusion partner, leading to pure active DRPs at milligram scale in many cases. These results open up new possibilities for the study and development of DRPs with therapeutic or biotechnological interest whose production was previously a limitation.

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Section: Discussionmentioning

confidence: 99%

High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli

et al. 2013

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“…Current protocols for production of heavy isotope and Se-Met labeled proteins using E. coli expression systems are mainly based on M9 minimal medium (Doublie 1997;Fiaux et al 2004;Leiting et al 1998;Mac et al 2006;Seo et al 2009;Zhao et al 2004) leading to very poor final biomass concentrations (OD600 of 1~2) and, consequently, low yields of labeled proteins. Some improvements have been obtained by optimizing the minimal medium (Jansson et al 1996) or by using autoinduction medium for the generation of 15 N and/or 13 C or Se-Met labeled proteins (Studier 2005;Sreenath et al 2005;Tyler et al 2005).…”

Section: Introductionmentioning

confidence: 99%

Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems

Nimtz

Rinas

2011

Appl Microbiol Biotechnol

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CitationOptimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems. 2011, 92 (4) AbstractGenerating sufficient quantities of labeled proteins represents a bottleneck in protein structure determination. A simple protocol for producing heavy isotope as well as selenomethionine (Se-Met) labeled proteins was developed using T7-based Escherichia coli expression systems. The protocol is applicable for generation of single, double and triple-labeled proteins ( 15 N, 13 C and 2 H) in shaker flask cultures. Label incorporation into the target protein reached 99% and 97% for 15 N and 13 C, respectively, and 75% of (non-exchangeable) hydrogen for 2 H labeling. The expression yields and final cell densities (OD600 ~ 16) were the same as for the production of non-labeled protein. This protocol is also applicable for Se-Met labeling, leading to Se-Met incorporation into the target protein of 70 or 90% using prototrophic or methionine auxotrophic E. coli strains, respectively.

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Merozoite surface protein 2 of Plasmodium falciparum: Expression, structure, dynamics, and fibril formation of the conserved N‐terminal domain

et al. 2007

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Merozoite surface protein 2 (MSP2) is a GPI-anchored protein on the surface of the merozoite stage of the malaria parasite Plasmodium falciparum. It is largely disordered in solution, but has a propensity to form amyloid-like fibrils under physiological conditions. The N-terminal conserved region (MSP2(1-25)) is part of the protease-resistant core of these fibrils. To investigate the structure and dynamics of this region, its ability to form fibrils, and the role of individual residues in these properties, we have developed a bacterial expression system that yields > or =10 mg of unlabeled or (15)N-labeled peptide per litre of culture. Two recombinant versions of MSP2(1-25), wild-type and a Y7A/Y16A mutant, have been produced. Detailed conformational analysis of the wild-type peptide and backbone (15)N relaxation data indicated that it contains beta-turn and nascent helical structures in the central and C-terminal regions. Residues 6-21 represent the most ordered region of the structure, although there is some flexibility around residues 8 and 9. The 10-residue sequence (MSP2(7-16)) (with two Tyr residues) was predicted to have a higher propensity for beta-aggregation than the 8-mer sequence (MSP2(8-15)), but there was no significant difference in conformation between MSP2(1-25) and [Y7A,Y16A]MSP2(1-25) and the rate of fibril formation was only slightly slower in the mutant. The peptide expression system described here will facilitate further mutational analyses to define the roles of individual residues in transient structural elements and fibril formation, and thus contribute to the further development of MSP2 as a malaria vaccine candidate.

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High yield expression and purification of isotopically labelled human endothelin-1 for use in NMR studies

Cited by 8 publications

References 18 publications

High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli

High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli

Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems

Merozoite surface protein 2 of Plasmodium falciparum: Expression, structure, dynamics, and fibril formation of the conserved N‐terminal domain

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