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1.
J Biomol NMR ; 71(4): 247-262, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29779067

RESUMO

We present protocols for high-level expression of isotope-labelled proteins in E. coli in cost-effective ways. This includes production of large amounts of unlabeled proteins and 13C-methyl methionine labeling in rich media, where yields of up to a gram of soluble protein per liter of culture are reached. Procedures for uniform isotope labeling of 2H, 13C and 15N using auto-induction or isopropyl-ß-D-1-thiogalactopyranoside-induction are described, with primary focus on minimal isotope consumption and high reproducibility of protein expression. These protocols are based on high cell-density fermentation, but the key procedures are easily transferred to shake flask cultures.


Assuntos
Marcação por Isótopo/economia , Ressonância Magnética Nuclear Biomolecular/métodos , Isótopos de Carbono , Deutério , Escherichia coli/metabolismo , Fermentação , Marcação por Isótopo/métodos , Metionina/análogos & derivados , Isótopos de Nitrogênio , Reprodutibilidade dos Testes
2.
J Biomol NMR ; 62(2): 169-77, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25893498

RESUMO

Auto-inducing media for protein expression offer many advantages like robust reproducibility, high yields of soluble protein and much reduced workload. Here, an auto-inducing medium for uniform isotope labelling of proteins with (15)N, (13)C and/or (2)H in E. coli is presented. So far, auto-inducing media have not found widespread application in the NMR field, because of the prohibitively high cost of labeled lactose, which is an essential ingredient of such media. Here, we propose using lactose that is only selectively labeled on the glucose moiety. It can be synthesized from inexpensive and readily available substrates: labeled glucose and unlabeled activated galactose. With this approach, uniformly isotope labeled proteins were expressed in unattended auto-inducing cultures with incorporation of (13)C, (15)N of 96.6% and (2)H, (15)N of 98.8%. With the present protocol, the NMR community could profit from the many advantages that auto-inducing media offer.


Assuntos
Marcação por Isótopo/métodos , Proteínas/química , Isótopos de Carbono/química , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Lactose/química , Isótopos de Nitrogênio/química , Ressonância Magnética Nuclear Biomolecular/métodos , Reprodutibilidade dos Testes
3.
PLoS One ; 12(4): e0174706, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28384226

RESUMO

RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRASG12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRASG12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRASG12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the in vitro assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery.


Assuntos
Lipídeos/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Espectroscopia de Ressonância Magnética , Prenilação
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