Untitled

Marley, Jonathan; Lu, Min; Bracken, Clay

doi:10.1023/a:1011254402785

Cited by 702 publications

(330 citation statements)

References 17 publications

Supporting

Mentioning

328

Contrasting

Order By: Relevance

“…Singly labeled 15 N-JadX-His 6 was prepared and purified. 47 At pH 7.6, there were few 1 H− 15 N HSQC cross peaks. To address this, the pH of the PBS was dropped to pH 6.6 to slow H−D exchange, resulting in an increase in the observable number of cross peaks ( Figure S18).…”

Section: ■ Introductionmentioning

confidence: 98%

JadX is a Disparate Natural Product Binding Protein

Robertson¹,

Forget²,

Martinez-Farina³

et al. 2016

J. Am. Chem. Soc.

View full text Add to dashboard Cite

Vous avez des questions? Nous pouvons vous aider. Pour communiquer directement avec un auteur, consultez la première page de la revue dans laquelle son article a été publié afin de trouver ses coordonnées. Si vous n'arrivez pas à les repérer, communiquez avec nous à PublicationsArchive-ArchivesPublications@nrc-cnrc.gc.ca. Questions? Contact the NRC Publications Archive team atPublicationsArchive-ArchivesPublications@nrc-cnrc.gc.ca. If you wish to email the authors directly, please see the first page of the publication for their contact information. NRC Publications Archive Archives des publications du CNRCThis publication could be one of several versions: author's original, accepted manuscript or the publisher's version. / La version de cette publication peut être l'une des suivantes : la version prépublication de l'auteur, la version acceptée du manuscrit ou la version de l'éditeur. NRC Publications Record / Notice d'Archives des publications de CNRC:http://nparc.cisti-icist.nrc-cnrc.gc.ca/eng/view/object/?id=1cef32c4-4c83-4b35-af83-2602ff6d1af3 http://nparc.cisti-icist.nrc-cnrc.gc.ca/fra/voir/objet/?id=1cef32c4-4c83-4b35-af83-2602ff6d1af3JadX is a Disparate Natural Product Binding Protein ABSTRACT: We report that JadX, a protein of previously undetermined function coded for in the jadomycin biosynthetic gene cluster of Streptomyces venezuelae ISP5230, affects both chloramphenicol and jadomycin production levels in blocked mutants. Characterization of recombinant JadX through protein−ligand interactions by chemical shift perturbation and WaterLOGSY NMR spectroscopy resulted in the observation of binding between JadX and a series of jadomycins and between JadX and chloramphenicol, another natural product produced by S. venezuelae ISP5230. These results suggest JadX to be an unusual class of natural product binding protein involved in binding structurally disparate natural products. The ability for JadX to bind two different natural products in vitro and the ability to affect production of these secondary metabolites in vivo suggest a potential role in regulation or signaling. This is the first example of functional characterization of these JadX-like proteins, and provides insight into a previously unobserved regulatory process.

show abstract

Section: ■ Introductionmentioning

confidence: 98%

JadX is a Disparate Natural Product Binding Protein

Robertson¹,

Forget²,

Martinez-Farina³

et al. 2016

J. Am. Chem. Soc.

View full text Add to dashboard Cite

show abstract

“…However, expression in minimal media, required in order to produce isotopically labeled proteins, did not provide pure MyrUSH3, presumably due to limited availability of carbon sources. This limitation was overcome by using the Marley method, which consists of generating cell mass in unlabeled rich media and subsequent transfer into labeled media just before induction 23. In that case we were able to obtain sufficient amounts of labeled proteins (95 %) to enable NMR measurement.…”

Section: Resultsmentioning

confidence: 99%

“…For the expression of isotopically labeled samples the protocol developed by Marley et al. was used:23 cells were first grown in LB to an OD 600 of 0.4 and were then centrifuged at 1000 g and 4 °C for 20 min. The pellet was resuspended in half the volume of minimal medium containing ammonium chloride and glucose.…”

Section: Methodsmentioning

confidence: 99%

N‐Lauroylation during the Expression of Recombinant N‐Myristoylated Proteins: Implications and Solutions

et al. 2015

View full text Add to dashboard Cite

Incorporation of myristic acid onto the N terminus of a protein is a crucial modification that promotes membrane binding and correct localization of important components of signaling pathways. Recombinant expression of N‐myristoylated proteins in Escherichia coli can be achieved by co‐expressing yeast N‐myristoyltransferase and supplementing the growth medium with myristic acid. However, undesired incorporation of the 12‐carbon fatty acid lauric acid can also occur (leading to heterogeneous samples), especially when the available carbon sources are scarce, as it is the case in minimal medium for the expression of isotopically enriched samples. By applying this method to the brain acid soluble protein 1 and the 1–185 N‐terminal region of c‐Src, we show the significant, and protein‐specific, differences in the membrane binding properties of lauroylated and myristoylated forms. We also present a robust strategy for obtaining lauryl‐free samples of myristoylated proteins in both rich and minimal media.

show abstract

“…Even after adaptation, cell densities reached in D 2 O did not increase above OD600 ~ 5 (Romanuka et al 2009;Paliy et al 2003). As alternative, cells can be grown in the presence of 75% D 2 O to avoid the strong inhibitory effect of pure D 2 O on growth and protein production, however, the reduced D 2 O content also reduces the deuteration efficiency of the target protein (Marley et al 2001). Furthermore, for all labeling methods, different protocols are currently employed, all with their unique stock solutions and procedures, making the production of highly labeled proteins tedious, more costly than necessary and most often the bottleneck of protein structure determination.…”

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

View full text Add to dashboard Cite

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.

show abstract

Untitled

Cited by 702 publications

References 17 publications

JadX is a Disparate Natural Product Binding Protein

JadX is a Disparate Natural Product Binding Protein

N‐Lauroylation during the Expression of Recombinant N‐Myristoylated Proteins: Implications and Solutions

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

Contact Info

Product

Resources

About