Separation of Copurifying GroEL from Glutathione-S-Transferase Fusion Proteins

Rohman, Mattias; Harrison‐Lavoie, Kimberly J.

doi:10.1006/prep.2000.1271

Cited by 41 publications

(36 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The addition of commonly used nonionic detergents (0.1% Triton X-100, 0.1 to 0.4% CHAPS, 1% OG), reducing agent (0.2 to 1 mM dithiothreitol), 5 to 10% glycerol, 10 to 15% acetonitrile to the buffer or high salt (500 to 700 mM NaCl) or low or high pH buffers (pH 4.5, pH 8.9, or pH 11) did not result in separation. The methods described in the literature for the removal of GroEL from GST fusion proteins (35,41) also failed in our case. However, we identified a commercial product, B-PER (Pierce Biotechnology Inc., Rockford, IL), to be highly effective in releasing GroEL from the affinity columnbound SV fusion proteins (Fig.…”

Section: Expression Of Sv Capsid Proteins In Bacteriamentioning

confidence: 61%

Development of an Enzyme Immunoassay for Detection of Sapovirus-Specific Antibodies and Its Application in a Study of Seroprevalence in Children

et al. 2006

View full text Add to dashboard Cite

Sapoviruses (SVs) are an important cause of acute pediatric gastroenteritis. Due to the lack of appropriate diagnostic methods, the epidemiology of SV-associated illness remains poorly understood. Baculovirus and Escherichia coli expression systems were evaluated for the development of antibody detection enzyme immunoassays (EIA). Age-related antibody prevalence in children was studied using the new EIA. Because of the low yield of the baculovirus system, the E. coli-expressed SV capsid proteins were used to develop the EIA. The antigenic specificities of the E. coli-expressed SV capsid proteins were demonstrated using hyperimmune antisera raised in animals and sera collected from patients. A high prevalence (>90%) of antibodies to both SV (strain Mex340) and norovirus (strain VA387) was observed in children involved in a birth cohort at 20 to 24 months of age; however, at 1 to 3 months of age, <25% of the children possessed anti-SV antibodies versus >90% with anti-NV antibodies. The E. coli-derived SV proteins are an excellent source of antigens for the EIA. SV infection is common in the first 2 years of life. The low prevalence of maternal antibodies detected in Mexican children against SVs in this study is unique and needs to be addressed in future studies.The family Caliciviridae consists of four genera, Norovirus (NV), Sapovirus (SV), Lagovirus, and Vesivirus, from which NV and SV cause disease mainly in humans and therefore are also referred to as human caliciviruses (HuCVs) (9). NVs are the leading cause of epidemic gastroenteritis, often causing large water-or food-borne outbreaks in all ages, while SVs are implicated mainly in pediatric gastroenteritis. In a study among Mexican children, we found that 19% of the sporadic acute gastroenteritis cases were associated with HuCVs and over a third of these were caused by SVs (8). Pang et al. reported 20% and 9% detection rates for NVs and SVs, respectively, in stool specimens of children with acute gastroenteritis in Finland (31,32). It is generally accepted that SV gastroenteritis is less severe than that of rotavirus or NV; however, SVs have been detected in 1 to 3% of stool specimens collected from children hospitalized with gastroenteritis, indicating that SVs can cause severe illness (34,38,47). Also, with the increasing awareness of SV gastroenteritis and improved diagnostic methods, SVs were recently implicated in several gastroenteritis outbreaks in adult populations (22,43).Because of the lack of a suitable animal model and tissue culture system for the propagation of HuCVs (5), the study of these pathogens has advanced slowly since the description of the prototype Norwalk and Sapporo viruses in the 1970s (3, 23). The cloning and genomic characterization of the Norwalk virus (14) and subsequently other HuCVs opened the way for the development of diagnostic techniques, such as reverse transcription-PCR (16) and enzyme immunoassays (EIA) utilizing recombinant virus-like particles (VLPs) (15, 17). These techniques proved to be superior to previously use...

show abstract

Section: Expression Of Sv Capsid Proteins In Bacteriamentioning

confidence: 61%

Development of an Enzyme Immunoassay for Detection of Sapovirus-Specific Antibodies and Its Application in a Study of Seroprevalence in Children

et al. 2006

View full text Add to dashboard Cite

show abstract

“…The supernatant was diluted 3-fold in 100 mM triethanolamine-HCl (pH 7.4) containing 150 mM NaCl, 20 mM MgCl 2 , 50 mM KCl, 1% CHAPS, 10 mM ATP, 1 mM DTT, and 1 mM PMSF (buffer A). Because the co-expressed GroEL (a gene product of pGro7) formed a complex with the NAPE-PLD-GST fusion protein, denatured proteins from bacterial lysate, which can bind to GroEL, were also added as described previously (33) except that the denatured proteins were precipitated with trichloroacetic acid. After incubation at 37°C for 20 min, the sample was centrifuged at 15,000 ϫ g for 30 min at 4°C, and to the resultant supernatant derived from 100 ml of the original culture, 2 ml of glutathione-Sepharose 4B beads pre-equilibrated with buffer A was added.…”

Section: Methodsmentioning

confidence: 99%

Functional Analysis of the Purified Anandamide-generating Phospholipase D as a Member of the Metallo-β-lactamase Family

Wang

Okamoto²,

Morishita³

et al. 2006

Journal of Biological Chemistry

107

View full text Add to dashboard Cite

In animal tissues, bioactive N-acylethanolamines including the endocannabinoid anandamide are formed from their corresponding N-acylphosphatidylethanolamines (NAPEs) by the catalysis of a specific phospholipase D (NAPE-PLD) that belongs to the metallo-␤-lactamase family. Despite its potential physiological importance, NAPE-PLD has not yet been characterized with a purified enzyme preparation. In the present study we expressed a recombinant NAPE-PLD in Escherichia coli and highly purified it. The purified enzyme was remarkably activated in a dose-dependent manner by millimolar concentrations of Mg 2؉ as well as Ca 2؉ and, hence, appeared to be constitutively active. The enzyme showed extremely high specificity for NAPEs among various glycerophospholipids but did not reveal obvious selectivity for different long chain or medium chain N-acyl species of NAPEs. These results suggested the ability of NAPE-PLD to degrade different NAPEs without damaging other membrane phospholipids. Metal analysis revealed the presence of catalytically important zinc in NAPE-PLD. In addition, sitedirected mutagenesis studies were addressed to several histidine and aspartic acid residues of NAPE-PLD that are highly conserved within the metallo-␤-lactamase family. Single mutations of Asp-147, His-185, His-187, Asp-189, His-190, His-253, Asp-284, and His-321 caused abolishment or remarkable reduction of the catalytic activity. Moreover, when six cysteine residues were individually mutated to serine, only C224S showed a considerably reduced activity. The activities of L207F and H380R found as single nucleotide polymorphisms were also low. Thus, NAPE-PLD appeared to function through a mechanism similar to those of the well characterized members of this family but play a unique role in the lipid metabolism of animal tissues.

show abstract

“…A single minor contaminating protein of approximately 64 kDa was detected in both preparations (data not shown). This contaminant was likely to be GroEL because this protein often copurifies with misfolded proteins in the E. coli host (27). R. capsulatus CbbR I and CbbR II were not insoluble in low-salt buffers as are a number of other LysR family regulatory proteins (4,9,20) and were used directly in DNA binding experiments.…”

Section: Construction Ofmentioning

confidence: 99%

Effector-Mediated Interaction of CbbR _I and CbbR _II Regulators with Target Sequences in Rhodobacter capsulatus

Dubbs

Tabita

2004

J Bacteriol

View full text Add to dashboard Cite

Rhodobacter capsulatus is a nonsulfur purple photosynthetic bacterium that possesses two cbb operons, cbb I and cbb II , encoding enzymes involved in the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway of carbon dioxide fixation (24, 25) (Fig. 1). The cbb I operon of R. capsulatus contains cbbL and cbbS, encoding the large and small subunits of form I ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO), respectively, as well as two genes, cbbQ and cbbO, of unknown function. Aside from cbbM, which encodes form II RubisCO, the cbb II operon also contains cbbF, encoding fructose-1,6/sedoheptulose-1,7-bisphosphatase; cbbP, encoding phosphoribulokinase; cbbT, encoding transketolase; cbbG, encoding glyceraldehyde 3-phosphate dehydrogenase; cbbA, encoding fructose-1,6-bisphosphate aldolase; cbbM, encoding the large subunit of form II RubisCO; cbbE, encoding ribulose-5-phosphate-3-epimerase; cbbZ, encoding 2-phosphoglycolate phosphatase; and cbbY, encoding an unknown function, as well as three unidentified open reading frames.Divergently transcribed from the cbb I and cbb II operons are cbbR I and cbbR I , respectively, which encode regulators that positively affect transcription of their cognate operons. R. capsulatus cbb I and cbb II are regulated independently, and their expression levels have been shown to vary depending on the growth conditions (13,14,24,25,29,43). The level of cbb I and cbb II expression is maximal under photoautotrophic conditions, when the CBB pathway is used to synthesize organic carbon from CO 2 to support growth and maintain the redox balance of the cell (18, 46). During photoheterotrophic growth (i.e., anaerobic growth conditions in the presence of an organic carbon source), cbb II expression is reduced, while cbb I is not expressed. Under aerobic chemoheterotrophic conditions, when CO 2 fixation is not needed, the level of cbb expression is lowest. Regulatory cross talk between the two operons also occurs, since inactivation of either of the two cbb operons in R. capsulatus leads to a compensatory increase in the expression of the remaining operon (25).While the RegB-RegA two-component regulatory system is also involved in derepressing both cbb I and cbb II operon expression under photoautotrophic growth conditions (43), CbbR I and CbbR II for the most part specifically regulate their cognate operons. However, there is some indication that, in the absence of CbbR II , CbbR I may cross regulate cbb II operon expression under photoheterotrophic conditions (43). In addition, partial expression of the cbb II operon occurs under photoautotrophic conditions in the complete absence of CbbR I

show abstract

Separation of Copurifying GroEL from Glutathione-S-Transferase Fusion Proteins

Cited by 41 publications

References 7 publications

Development of an Enzyme Immunoassay for Detection of Sapovirus-Specific Antibodies and Its Application in a Study of Seroprevalence in Children

Development of an Enzyme Immunoassay for Detection of Sapovirus-Specific Antibodies and Its Application in a Study of Seroprevalence in Children

Functional Analysis of the Purified Anandamide-generating Phospholipase D as a Member of the Metallo-β-lactamase Family

Effector-Mediated Interaction of CbbR _I and CbbR _II Regulators with Target Sequences in Rhodobacter capsulatus

Contact Info

Product

Resources

About

Separation of Copurifying GroEL from Glutathione-S-Transferase Fusion Proteins

Cited by 41 publications

References 7 publications

Development of an Enzyme Immunoassay for Detection of Sapovirus-Specific Antibodies and Its Application in a Study of Seroprevalence in Children

Development of an Enzyme Immunoassay for Detection of Sapovirus-Specific Antibodies and Its Application in a Study of Seroprevalence in Children

Functional Analysis of the Purified Anandamide-generating Phospholipase D as a Member of the Metallo-β-lactamase Family

Effector-Mediated Interaction of CbbR I and CbbR II Regulators with Target Sequences in Rhodobacter capsulatus

Contact Info

Product

Resources

About

Effector-Mediated Interaction of CbbR _I and CbbR _II Regulators with Target Sequences in Rhodobacter capsulatus