Selectively labeling the heterologous protein in Escherichia coli for NMR studies: a strategy to speed up NMR spectroscopy.
Nuclear magnetic resonance is an important tool for high-resolution structural studies of proteins. It demands high protein concentration and high purity; however, the expression of proteins at high levels often leads to protein aggregation and the protein purification step can correspond to a high percentage of the overall time in the structural determination process. In the present article we show that the step of sample optimization can be simplified by selective labeling the heterologous protein expressed in Escherichia coli by the use of rifampicin. Yeast thioredoxin and a coix transcription factor Opaque 2 leucine zipper (LZ) were used to show the effectiveness of the protocol. The (1)H/(15)N heteronuclear correlation two-dimensional NMR spectrum (HMQC) of the selective (15)N-labeled thioredoxin without any purification is remarkably similar to the spectrum of the purified protein. The method has high yields and a good (1)H/(15)N HMQC spectrum can be obtained with 50 ml of M9 growth medium. Opaque 2 LZ, a difficult protein due to the lower expression level and high hydrophobicity, was also probed. The (15)N-edited spectrum of Opaque 2 LZ showed only the resonances of the protein of heterologous expression (Opaque 2 LZ) while the (1)H spectrum shows several other resonances from other proteins of the cell lysate. The demand for a fast methodology for structural determination is increasing with the advent of genome/proteome projects. Selective labeling the heterologous protein can speed up NMR structural studies as well as NMR-based drug screening. This methodology is especially effective for difficult proteins such as hydrophobic transcription factors, membrane proteins, and others.
Almeida
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FC
Departamento de Bioquímica Médica, Centro Nacional de Ressonância Magnética Nuclear, Universidade Federal do Rio de Janeiro--UFRJ, Cidade Universitária, RJ, 21941-590, Brazil. famieda@cnrmn.bioqmed.ufrj.br
Amorim
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GC
Moreau
V H
VH
Sousa
V O
VO
Creazola
A T
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Américo
T A
TA
Pais
A P
AP
Leite
A
A
Netto
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LE
Giordano
R J
RJ
Valente
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AP
D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry*; D016350:Leucine Zippers; D019906:Nuclear Magnetic Resonance, Biomolecular; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry*; D012293:Rifampin / Q000737:chemistry / Q000494:pharmacology; D013879:Thioredoxins / Q000096:biosynthesis / Q000737:chemistry*; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D010940:Plant Proteins; D014157:Transcription Factors; C067402:opaque-2 protein, Zea mays; D013879:Thioredoxins; D012293:Rifampin