Expression of a Recombinant Toxoplasma gondii ROP2 Fragment as a Fusion Protein in Bacteria Circumvents Insolubility and Proteolytic Degradation

Jacquet, Alain; Daminet,; Haumont, Michèle; Garcia, Lida; Chaudoir, S; Bollen, Alex; Biemans, Ralph

doi:10.1006/prep.1999.1150

Cited by 39 publications

(22 citation statements)

References 20 publications

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“…In addition, ROP2 and its family are potent humoral and T-cell antigens (Saavedra et al, 1996;Jacquet et al, 1999). It also carries B-cell epitopes because antibodies against the ROP2 antigen are present in Journal of Cell Science 116 (11) more than 85% of T. gondii-seropositive individuals (Van Gelder et al, 1993).…”

Section: Discussionmentioning

confidence: 99%

Pleiotropic effect due to targeted depletion of secretory rhoptry protein ROP2 inToxoplasma gondii

Nakaar

Ngô

Aaronson

et al. 2003

Journal of Cell Science

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Long after their discovery, the function and biogenesis of rhoptries remain enigmatic. In Apicomplexan parasites, these organelles discharge and their contents are exocytosed at the time of host cell invasion, and are thus proposed to play an essential role in establishing the parasitophorous vacuole. In Toxoplasma gondii, ROP2 is suspected to serve as the molecular link between host cell mitochondria and parasitophorous vacuole membrane. In this study we addressed the function of ROP2. Targeted depletion of ROP2 using a ribozyme-modified antisense RNA strategy resulted in multiple effects on parasite morphology because of a disruption in the formation of mature rhoptries, and an arrest in cytokinesis. The association of host cell mitochondria with the parasitophorous vacuole membrane was abolished and the ROP2-deficient parasites had a reduced uptake of sterol from the host cell. Furthermore, these parasites invaded human fibroblasts poorly and had markedly attenuated virulence in mice. We conclude that rhoptry discharge, and in particular release of ROP2, are essential for parasite invasion, replication and host cell-parasite interaction.

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Section: Discussionmentioning

confidence: 99%

Pleiotropic effect due to targeted depletion of secretory rhoptry protein ROP2 inToxoplasma gondii

Nakaar

Ngô

Aaronson

et al. 2003

Journal of Cell Science

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“…The E. coli expression system lacks many codons essentially requires for the expression of eukaryotic protein and also lack post translational modification machinery for biological activity and some other complex proteins containing multiple disulfide bonds [31,32,33]. In addition to codon bias and lack of post translational modification machinery, many proteins expressed in E. coli got accumulated intracellular as inactive inclusion bodies [34,35]. Expressed protein in the form of inclusion bodies further needs protein renaturation and refolding to retain biological activity, all these transformations are often complicated and costly while running denaturation and refolding process [36].…”

Section: Complications With Conventional Expression Systemmentioning

confidence: 99%

New Generation Expression Host System- Aiming high for commercial production of recombinant protein

Verma¹

2012

IOSJPBS

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“…In contrast to these multicomponent protein folding/disaggregation systems, other proteins exert the molecular chaperone function from within a much smaller ensemble or even without an obligatory cofactor (10). These simpler molecular chaperones include peptidyl prolyl cis-trans isomerase, thioredoxin, and disulfide isomerase dsbA (8,(11)(12)(13) and have been fused to target proteins to act as solubility enhancers. The simple operation and minimal strain manipulation make such molecular chaperones particularly attractive in synthesizing recombinant proteins.…”

mentioning

confidence: 99%

Protein Interaction Module–assisted Function X (PIMAX) Approach to Producing Challenging Proteins Including Hyperphosphorylated Tau and Active CDK5/p25 Kinase Complex

Sui

et al. 2015

Molecular & Cellular Proteomics

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Many biomedically critical proteins are underrepresented in proteomics and biochemical studies because of the difficulty of their production in Escherichia coli. These proteins might possess posttranslational modifications vital to their functions, tend to misfold and be partitioned into bacterial inclusion bodies, or act only in a stoichiometric dimeric complex. Successful production of these proteins requires efficient interaction between these proteins and a specific "facilitator," such as a protein-modifying enzyme, a molecular chaperone, or a natural physical partner within the dimeric complex. Here we report the design and application of a protein interaction moduleassisted function X (PIMAX) system that effectively overcomes these hurdles. By fusing two proteins of interest to a pair of well-studied protein-protein interaction modules, we were able to potentiate the association of these two proteins, resulting in successful production of an enzymatically active cyclin-dependent kinase complex and hyperphosphorylated tau protein, which is intimately linked to Alzheimer disease. Furthermore, using tau isoforms quantitatively phosphorylated by GSK-3␤ and CDK5 kinases via PIMAX, we demonstrated the hyperphosphorylation-stimulated tau oligomerization in vitro, paving the way for new Alzheimer disease drug discoveries. Vectors for PIMAX can be easily modified to meet the needs of different applications. This approach thus provides a convenient and modular suite with broad implications for proteomics and biomedical research. Molecular & Cellular Proteomics

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Expression of a Recombinant Toxoplasma gondii ROP2 Fragment as a Fusion Protein in Bacteria Circumvents Insolubility and Proteolytic Degradation

Cited by 39 publications

References 20 publications

Pleiotropic effect due to targeted depletion of secretory rhoptry protein ROP2 inToxoplasma gondii

Pleiotropic effect due to targeted depletion of secretory rhoptry protein ROP2 inToxoplasma gondii

New Generation Expression Host System- Aiming high for commercial production of recombinant protein

Protein Interaction Module–assisted Function X (PIMAX) Approach to Producing Challenging Proteins Including Hyperphosphorylated Tau and Active CDK5/p25 Kinase Complex

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