A method for the separation of GST fusion proteins from co-purifying GroEL

Thain, Alison; Gaston, Kevin; Jenkins, Owen; Clarke, Anthony R.

doi:10.1016/s0168-9525(96)90022-0

Cited by 55 publications

(41 citation statements)

References 5 publications

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“…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: 65%

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

et al. 2006

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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...

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Section: Expression Of Sv Capsid Proteins In Bacteriamentioning

confidence: 65%

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

et al. 2006

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“…Moreover, attempts to use an engineered E. coli strain (43) (a kind gift from Prof. Ulrich Hartl's laboratory, Max Planck Institute for Biochemistry, Munich, Germany) carrying the GroEL/ES operon under an arabinose-inducible promoter, which allows tuned regulation of the expression levels of these chaperones, failed to produce the hASNase1 protein (data not shown). Ultimately, we managed to remove the co-purified chaperone by including in the washing buffer the natural binding partner of this protein, GroES, at ϳ10-fold excess over GroEL (44). It appears reasonable to assume that the presence of ATP-Mg 2ϩ -GroES weakened the binding between GroEL and hASNase1, thus facilitat-ing their separation upon exhaustive washing (Fig.…”

Section: Resultsmentioning

confidence: 99%

Human 60-kDa Lysophospholipase Contains an N-terminal l-Asparaginase Domain That Is Allosterically Regulated by l-Asparagine

Karamitros

Konrad

2014

Journal of Biological Chemistry

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Background:The 60-kDa human lysophospholipase comprises an N-terminal domain with predicted, yet uncharacterized L-asparaginase activity and a C-terminal ankyrin repeat-like domain. Results:The N-terminal domain, termed hASNase1, was identified as a functional structural unit possessing catalytic activity. Conclusion: hASNase1 is an allosterically regulated bacterial-type cytoplasmic L-asparaginase. Significance: Domains of multifunctional human proteins harbor homologs of prokaryotic enzymes displaying similar structural and kinetic features.

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“…Protein was extracted and purified as described previously (29). Copurifying, heat shock bacterial chaperonin GroEL (ϳ65 kDa) was removed from VP5* and VP5*D308A/G309A by using ATP and partner chaperonin GroES (Sigma), as described previously (52). As complete removal of GroEL rendered VP5* insoluble, GroEL was not removed from VP5* for further experiments.…”

Section: Methodsmentioning

confidence: 99%

Integrin-Using Rotaviruses Bind α2β1 Integrin α2 I Domain via VP4 DGE Sequence and Recognize αXβ2 and αVβ3 by Using VP7 during Cell Entry

Graham

Halász

Tan

et al. 2003

J Virol

143

186

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Integrins ␣2␤1, ␣X␤2, and ␣V␤3 have been implicated in rotavirus cell attachment and entry. The virus spike protein VP4 contains the ␣2␤1 ligand sequence DGE at amino acid positions 308 to 310, and the outer capsid protein VP7 contains the ␣X␤2 ligand sequence GPR. To determine the viral proteins and sequences involved and to define the roles of ␣2␤1, ␣X␤2, and ␣V␤3, we analyzed the ability of rotaviruses and their reassortants to use these integrins for cell binding and infection and the effect of peptides DGEA and GPRP on these events. Many laboratory-adapted human, monkey, and bovine viruses used integrins, whereas all porcine viruses were integrin independent. The integrin-using rotavirus strains each interacted with all three integrins. Integrin usage related to VP4 serotype independently of sialic acid usage. Analysis of rotavirus reassortants and assays of virus binding and infectivity in integrin-transfected cells showed that VP4 bound ␣2␤1, and VP7 interacted with ␣X␤2 and ␣V␤3 at a postbinding stage. DGEA inhibited rotavirus binding to ␣2␤1 and infectivity, whereas GPRP binding to ␣X␤2 inhibited infectivity but not binding. The truncated VP5* subunit of VP4, expressed as a glutathione S-transferase fusion protein, bound the expressed ␣2 I domain. Alanine mutagenesis of D308 and G309 in VP5* eliminated VP5* binding to the ␣2 I domain. In a novel process, integrin-using viruses bind the ␣2 I domain of ␣2␤1 via DGE in VP4 and interact with ␣X␤2 (via GPR) and ␣V␤3 by using VP7 to facilitate cell entry and infection.Rotaviruses are leading causes of acute gastroenteritis in human infants and young animals. Their restricted tropism suggests that very specific virus-host cell interactions are necessary to establish infection. The viral spike protein VP4, the major cell attachment protein, is cleaved by trypsin for enhanced infectivity into two subunits, VP5* (60 kDa) and VP8* (28 kDa). VP4 and the major outer capsid protein VP7 independently define serotype specificities. The inner capsid protein VP6 contains group-specific antigenic determinants. Reassortant rotaviruses containing combinations of the 11 double-stranded RNA genes from two parental viruses can be generated (27) which occasionally show unexpected phenotypes due to VP4-VP7 interactions (43).Integrins ␣2␤1, ␣X␤2, and ␣4␤1 were implicated in group A rotavirus cell attachment and entry (11, 23), and ␣V␤3 was proposed to mediate rotavirus cell entry (20). Integrin usage by rotaviruses was discovered when VP5* was shown to contain the type I collagen-derived, ␣2␤1 ligand sequence DGE at amino acids 308 to 310, and the fibrinogen-derived ␣X␤2 integrin ligand sequence GPR was identified in VP7 at amino acids 253 to 255 (11). Peptides containing these viral integrin ligand sequences (GPRP and RDGEE), monoclonal antibodies (MAbs) to ␣2␤1 and MAbs to ␤2, inhibited the infection of monolayers of MA104 and Caco-2 cells by simian rotavirus SA11 and/or human rotavirus RV-5 by 30 to 90% in additive fashion (9, 11). Infectivity blockade in MA104 cell monolayers...

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A method for the separation of GST fusion proteins from co-purifying GroEL

Cited by 55 publications

References 5 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

Human 60-kDa Lysophospholipase Contains an N-terminal l-Asparaginase Domain That Is Allosterically Regulated by l-Asparagine

Integrin-Using Rotaviruses Bind α2β1 Integrin α2 I Domain via VP4 DGE Sequence and Recognize αXβ2 and αVβ3 by Using VP7 during Cell Entry

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