Purification of recombinant human N-Acetyltransferase type 1 (NAT1) expressed in E. Coli and characterization of its potential role in folate metabolism

1995 ◽  
Vol 49 (12) ◽  
pp. 1759-1767 ◽  
Author(s):  
A. Ward ◽  
M.J. Summers ◽  
E. Sim
Keyword(s):  
Potential Role ◽  
Folate Metabolism ◽  
E Coli

Characterization of an immunogenic cellulase secreted by Cryptococcus pathogens

2020 ◽  
Vol 58 (8) ◽  
pp. 1138-1148
Author(s):  
Angelina Midiri ◽  
Giuseppe Mancuso ◽  
Germana Lentini ◽  
Agata Famà ◽  
Roberta Galbo ◽  
...  
Keyword(s):  
Species Complex ◽  
Delayed Type Hypersensitivity ◽  
T Helper ◽  
Terminal Sequence ◽  
E Coli ◽  
Culture Supernatants ◽  
Highly Virulent ◽  
Potential Use

Abstract Members of the C. neoformans/C. gattiii species complex are an important cause of serious humans infections, including meningoencephalitis. We describe here a 45 kDa extracellular cellulase purified from culture supernatants of C. neoformans var. neoformans. The N-terminal sequence obtained from the purified protein was used to isolate a clone containing the full-length coding sequence from a C. neoformans var. neoformans (strain B-3501A) cDNA library. Bioinformatics analysis indicated that this gene is present, with variable homology, in all sequenced genomes of the C. neoformans/C. gattii species complex. The cDNA clone was used to produce a recombinant 45 kDa protein in E. coli that displayed the ability to convert carboxymethyl cellulose and was therefore designated as NG-Case (standing for Neoformans Gattii Cellulase). To explore its potential use as a vaccine candidate, the recombinant protein was used to immunize mice and was found capable of inducing T helper type 1 responses and delayed-type hypersensitivity reactions, but not immune protection against a highly virulent C. neoformans var grubii strain. These data may be useful to better understand the mechanisms underlying the ability C. neoformans/C. gattii to colonize plant habitats and to interact with the human host during infection.

scholarly journals Cloning and Characterization of a Gene Cluster for Cyclododecanone Oxidation in Rhodococcus ruber SC1

2001 ◽  
Vol 183 (21) ◽  
pp. 6478-6486 ◽  
Author(s):  
Kristy Kostichka ◽  
Stuart M. Thomas ◽  
Katharine J. Gibson ◽  
Vasantha Nagarajan ◽  
Qiong Cheng
Keyword(s):  
Gene Cluster ◽  
Dicarboxylic Acids ◽  
Rhodococcus Ruber ◽  
Cyclic Ketones ◽  
E Coli ◽  
Flavin Monooxygenase ◽  
Dodecanedioic Acid ◽  
Cyclic Compounds

ABSTRACT Biological oxidation of cyclic ketones normally results in formation of the corresponding dicarboxylic acids, which are further metabolized in the cell. Rhodococcus ruber strain SC1 was isolated from an industrial wastewater bioreactor that was able to utilize cyclododecanone as the sole carbon source. A reverse genetic approach was used to isolate a 10-kb gene cluster containing all genes required for oxidative conversion of cyclododecanone to 1,12-dodecanedioic acid (DDDA). The genes required for cyclododecanone oxidation were only marginally similar to the analogous genes for cyclohexanone oxidation. The biochemical function of the enzymes encoded on the 10-kb gene cluster, the flavin monooxygenase, the lactone hydrolase, the alcohol dehydrogenase, and the aldehyde dehydrogenase, was determined in Escherichia coli based on the ability to convert cyclododecanone. Recombinant E. colistrains grown in the presence of cyclododecanone accumulated lauryl lactone, 12-hydroxylauric acid, and/or DDDA depending on the genes cloned. The cyclododecanone monooxygenase is a type 1 Baeyer-Villiger flavin monooxygenase (FAD as cofactor) and exhibited substrate specificity towards long-chain cyclic ketones (C11 to C15), which is different from the specificity of cyclohexanone monooxygenase favoring short-chain cyclic compounds (C5 to C7).

Expression and characterization of a metalloprotease from aVibrio parahaemolyticusisolate

2007 ◽  
Vol 53 (10) ◽  
pp. 1168-1173 ◽  
Author(s):  
Xiaoyan Luan ◽  
Jixiang Chen ◽  
Xiao-hua Zhang ◽  
Yun Li ◽  
Guobin Hu
Keyword(s):  
Potential Role ◽  
Maximum Activity ◽  
Affinity Column ◽  
Putative Virulence Factor ◽  
E Coli ◽  
Metal Affinity ◽  
Zinc Metalloprotease ◽  
Gill Cells ◽  
Host Infection

The extracellular zinc metalloprotease from Vibrio parahaemolyticus (VPM) is a putative virulence factor for host infection. It is synthesized from the vpm gene of V. parahaemolyticus as a polypeptide of 814 amino acids with an estimated molecular mass of 89 833 Da, containing a zinc metalloprotease HEXXH consensus motif. To investigate the enzymatic properties of V. parahaemolyticus metalloprotease, the mature vpm gene was overexpressed in Escherichia coli , and the recombinant protein (rVPM) was purified by a His-binding metal affinity column (>95% purity). The activity of the recombinant protease produced in E. coli was examined by gelatin activity staining and proteolytic activity assays using gelatin and azocasein as substrates. rVPM showed maximum activity at about 37 °C and pH 8. The cytotoxicity against flounder gill cells and fish pathogenicity indicated a potential role in pathogenesis.

scholarly journals Characterization of the Escherichia coliAF/R1 Pilus Operon: Novel Genes Necessary for Transcriptional Regulation and for Pilus-Mediated Adherence

1999 ◽  
Vol 67 (5) ◽  
pp. 2292-2298 ◽  
Author(s):  
J. Robert Cantey ◽  
R. K. Blake ◽  
J. R. Williford ◽  
Steve L. Moseley
Keyword(s):  
Transcriptional Activation ◽  
Rabbit Model ◽  
M Cell ◽  
Ileal Mucosa ◽  
E Coli ◽  
Type 1 Pili ◽  
Structural Subunit ◽  
And Function

ABSTRACT We isolated the genetic determinant of AF/R1 pilus production in attaching/effacing Escherichia coli RDEC-1 and identified seven genes required for pilus expression and function. DNA sequence analysis of the structural subunit gene afrA corrected an error in the published sequence and extended homology with the F18 pilus subunit of pig edema E. coli strains. AfrB and AfrC, encoded downstream from AfrA, were required for pilus expression. AfrB was related to the usher protein PefC of Salmonella typhimurium plasmid-encoded fimbriae, and AfrC was related to PefD, a chaperone protein. AfrD and AfrE, encoded downstream from AfrC, were not necessary for the expression of AF/R1 pili but were required for ileal adherence as assayed by ileal brush border aggregation. Thus, the adhesive subunit of the AF/R1 pilus is distinct from the structural subunit, as is the case for Pap pili and type 1 pili. AfrD was related to FedE of the F18 fimbrial operon of the E. coli strain that causes edema disease in pigs. AfrE was a novel protein. AfrR and AfrS are encoded upstream from AfrA, in the opposite orientation. AfrR is related to the AraC family of transcriptional regulators, and AfrR and AfrS interact to function in a novel mode of transcriptional activation of afrA. AF/R1 pili mediate the adherence to Peyer’s patch M cells, ileal mucosa, and colonic mucosa in a rabbit model of diarrhea caused by enteropathogenic E. coli. Our observations will facilitate the further study of the phenomena of M-cell adherence.

Identification and characterization of E. coli type-1 pilus tip adhesion protein

Nature ◽  
1988 ◽  
Vol 332 (6161) ◽  
pp. 265-268 ◽  
Author(s):  
Mark S. Hanson ◽  
Charles C. Brinton
Keyword(s):  
Adhesion Protein ◽  
E Coli ◽  
Identification And Characterization

436-P: Characterization of Autoimmunity in the Acceleration of Atherosclerosis in Type 1 Diabetes

Diabetes ◽  
2020 ◽  
Vol 69 (Supplement 1) ◽  
pp. 436-P
Author(s):  
KYOUNGMIN PARK ◽  
QIAN LI ◽  
HYUNSEOK PARK ◽  
RONALD ST-LOUIS ◽  
JIALIN FU ◽  
...  
Keyword(s):  
Type 1 Diabetes

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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