Use of lactose to induce expression of soluble NifA protein domains ofHerbaspirillum seropedicaeinEscherichia coli

Overexpression and purification are procedures used to allow functional and structural characterization of proteins. Many overexpressed proteins are partially or completely insoluble, and can not be easily purified. The NifA protein is an enhancer-binding protein involved in activating the expression of nif and some fix genes. The NifA protein from many organisms is usually insoluble when over-expressed, and therefore difficult to work with in vitro. In this work we have overexpressed the central+C-terminal and the central domains of the Herbaspirrilum seropedicae NifA protein in an Escherichia coli background. Expression was induced with either IPTG or lactose. The data showed that induction with lactose promoted a significantly higher percentage of these proteins in the soluble fraction than with IPTG. This probably reflects a slower kinetics of induction by lactose.Key words: Herbaspirillum seropedicae, NifA protein, transcriptional activator, nitrogen fixation, protein expression.

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Characterization of a mutation affecting the function of Escherichia coli folylpolyglutamate synthetase-dihydrofolate synthetase and further mutations produced in vitro at the same locus.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)54871-7 ◽

1991 ◽

Vol 266 (30) ◽

pp. 19925-19929

Author(s):

K. Keshavjee ◽

C. Pyne ◽

A.L. Bognar

Keyword(s):

Escherichia Coli ◽

Folylpolyglutamate Synthetase

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In vitro transcription of the supB-E tRNA operon of Escherichia coli. Characterization of transcription products.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)33940-1 ◽

1982 ◽

Vol 257 (18) ◽

pp. 11113-11120 ◽

Cited By ~ 2

Author(s):

N Nakajima ◽

H Ozeki ◽

Y Shimura

Keyword(s):

Escherichia Coli ◽

In Vitro Transcription

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Characterization of a Dye-Decolorizing Peroxidase from Irpex lacteus Expressed in Escherichia coli: An Enzyme with Wide Substrate Specificity Able to Transform Lignosulfonates

Journal of Fungi ◽

10.3390/jof7050325 ◽

2021 ◽

Vol 7 (5) ◽

pp. 325

Author(s):

Laura Isabel de de Eugenio ◽

Rosa Peces-Pérez ◽

Dolores Linde ◽

Alicia Prieto ◽

Jorge Barriuso ◽

...

Keyword(s):

Escherichia Coli ◽

Inclusion Bodies ◽

Veratryl Alcohol ◽

Dye Decolorization ◽

Irpex Lacteus ◽

Type D ◽

Lignin Peroxidases

A dye-decolorizing peroxidase (DyP) from Irpex lacteus was cloned and heterologously expressed as inclusion bodies in Escherichia coli. The protein was purified in one chromatographic step after its in vitro activation. It was active on ABTS, 2,6-dimethoxyphenol (DMP), and anthraquinoid and azo dyes as reported for other fungal DyPs, but it was also able to oxidize Mn2+ (as manganese peroxidases and versatile peroxidases) and veratryl alcohol (VA) (as lignin peroxidases and versatile peroxidases). This corroborated that I. lacteus DyPs are the only enzymes able to oxidize high redox potential dyes, VA and Mn+2. Phylogenetic analysis grouped this enzyme with other type D-DyPs from basidiomycetes. In addition to its interest for dye decolorization, the results of the transformation of softwood and hardwood lignosulfonates suggest a putative biological role of this enzyme in the degradation of phenolic lignin.

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Characterization of fluoroquinolone-resistant mutants of escherichia coli selected in vitro.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.38.6.1284 ◽

1994 ◽

Vol 38 (6) ◽

pp. 1284-1291 ◽

Cited By ~ 60

Author(s):

P Heisig ◽

R Tschorny

Keyword(s):

Escherichia Coli ◽

Resistant Mutants

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Characterization of the Two Mycobacterium tuberculosis recA Promoters

Journal of Bacteriology ◽

10.1128/jb.185.20.6005-6015.2003 ◽

2003 ◽

Vol 185 (20) ◽

pp. 6005-6015 ◽

Cited By ~ 21

Author(s):

Krishna K. Gopaul ◽

Patricia C. Brooks ◽

Jean-François Prost ◽

Elaine O. Davis

Keyword(s):

Escherichia Coli ◽

Dna Damage ◽

Mycobacterium Tuberculosis ◽

Promoter Activity ◽

The Other ◽

Expression Level ◽

Promoter Elements ◽

Kinetics Of

ABSTRACT The recA gene of Mycobacterium tuberculosis is unusual in that it is expressed from two promoters, one of which, P1, is DNA damage inducible independently of LexA and RecA, while the other, P2, is regulated by LexA in the classical way (E. O. Davis, B. Springer, K. K. Gopaul, K. G. Papavinasasundaram, P. Sander, and E. C. Böttger, Mol. Microbiol. 46:791-800, 2002). In this study we characterized these two promoters in more detail. Firstly, we localized the promoter elements for each of the promoters, and in so doing we identified a mutation in each promoter which eliminates promoter activity. Interestingly, a motif with similarity to Escherichia coli σ70 −35 elements but located much closer to the −10 element is important for optimal expression of P1, whereas the sequence at the −35 location is not. Secondly, we found that the sequences flanking the promoters can have a profound effect on the expression level directed by each of the promoters. Finally, we examined the contribution of each of the promoters to recA expression and compared their kinetics of induction following DNA damage.

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Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.

Acta Biochimica Polonica ◽

10.18388/abp.2003_3623 ◽

2003 ◽

Vol 50 (4) ◽

pp. 909-920 ◽

Cited By ~ 4

Author(s):

Iwona K Kolasa ◽

Tomasz Łoziński ◽

Kazimierz L Wierzchowski

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Transcription Initiation ◽

Specific Interaction ◽

Structural Data ◽

Structural Morphology ◽

Promoter Dna ◽

Sigma Subunit ◽

Kinetics Of

A-tracts in DNA due to their structural morphology distinctly different from the canonical B-DNA form play an important role in specific recognition of bacterial upstream promoter elements by the carboxyl terminal domain of RNA polymerase alpha subunit and, in turn, in the process of transcription initiation. They are only rarely found in the spacer promoter regions separating the -35 and -10 recognition hexamers. At present, the nature of the protein-DNA contacts formed between RNA polymerase and promoter DNA in transcription initiation can only be inferred from low resolution structural data and mutational and crosslinking experiments. To probe these contacts further, we constructed derivatives of a model Pa promoter bearing in the spacer region one or two An (n = 5 or 6) tracts, in phase with the DNA helical repeat, and studied the effects of thereby induced perturbation of promoter DNA structure on the kinetics of open complex (RPo) formation in vitro by Escherichia coli RNA polymerase. We found that the overall second-order rate constant ka of RPo formation, relative to that at the control promoter, was strongly reduced by one to two orders of magnitude only when the A-tracts were located in the nontemplate strand. A particularly strong 30-fold down effect on ka was exerted by nontemplate A-tracts in the -10 extended promoter region, where an involvement of nontemplate TG (-14, -15) sequence in a specific interaction with region 3 of sigma-subunit is postulated. A-tracts in the latter location caused also 3-fold slower isomerization of the first closed transcription complex into the intermediate one that precedes formation of RPo, and led to two-fold faster dissociation of the latter. All these findings are discussed in relation to recent structural and kinetic models of RPo formation.

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