Dictyostelium discoideum as Expression Host: Isotopic Labeling of a Recombinant Glycoprotein for NMR Studies

Cubeddu, Liza; Moss, Catherine X.; Swarbrick, James; Gooley, Andrew A.; Williams, Keith L.; Curmi, Paul M. G.; Slade, Martin B.; Mabbutt, Bridget C.

doi:10.1006/prep.2000.1269

Cited by 21 publications

(15 citation statements)

References 25 publications

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“…[ 15 N, 13 C]-labelled rPsA was directly secreted by D. discoideum growing in MES buffer with labelled bacterial cells, as previously described (Cubeddu et al 2000). Following purification, NMR samples of [ 15 N, 13 C]-rPsA were prepared at approximately 1 mM in 25 mM acetate buffer, pH 6.0 in either 10 or 100% D 2 O. NMR experiments were performed at 35°C on a Bruker AVANCE DRX 600 spectrometer equipped with a room temperature probe.…”

Section: Methods and Experimentsmentioning

confidence: 99%

“…In order to pursue structural studies of PsA, we engineered a secreted form of 15.5 kDa truncated to contain only the globular domain (residues 1-91) attached to its linker segment (residues 92-121) (Cubeddu et al 2000). Within the linker peptide, this recombinant glycoprotein is further simplified, as the 13 O-linked glycosylation sites are modified by single N-acetyl glucosamine units only.…”

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confidence: 99%

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NMR assignment of prespore specific antigen—a cell surface adhesion glycoprotein from Dictyostelium discoideum

et al. 2008

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Presopore-specific antigen (PsA) is a cell surface glycoprotein of the cellular slime mould Dictyostelium discoidum implicated in cell adhesion. The (15)N, (13)C and (1)H chemical shift assignments of PsA were determined from multidimensional, multinuclear NMR experiments. Resonance assignments have been made for both the N-terminal globular domain and its attached O-glycosylated PTVT linker motif.

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Section: Methods and Experimentsmentioning

confidence: 99%

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confidence: 99%

NMR assignment of prespore specific antigen—a cell surface adhesion glycoprotein from Dictyostelium discoideum

et al. 2008

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“…It has a post-translational modification mechanism which is comparable to that of mammalian cells (Varki et al 1999). Several heterologous recombinant proteins with potential medical values have already been expressed successfully using this system (Dingermann et al 1991;Reymond et al 1995;Emslie et al 1996;Heikoop et al 1998;Cubeddu et al 2000;Asgari et al 2001;Lu et al 2004a). Compared to other microbial expression systems, D. discoideum system is attractive in scale, economics, and ease of manipulation and production of functional protein.…”

Section: Introductionmentioning

confidence: 93%

Efficient expression and primary purification of 6-his tagged human Fas ligand in Dictyostelium discoideum

Chen

et al. 2007

Biotechnol Lett

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Human Fas ligand (hFasL) is a member of the tumor necrosis factor (TNF) family with many medical interests. To produce this protein efficiently, an improved vector which could express the recombinant hFasL protein with a 6-his tag at its C-terminal was constructed. The new vector was transformed into Dictyostelium discoideum AX3 which then produced 157 microg hFasL l(-1). Using one-step Ni-affinity chromatography, it was purified with a recovery of 92% and purity of 91%.

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“…• Stable isotope or selenomethionine labeling is easier with cell-free systems than with fungal or insect cell systems [4][5][6]. • Cell-free systems permit the production of proteins that undergo proteolysis in cells [7,8] or aggregate in inclusion bodies [9].…”

Section: Expert Opinion and Five-year Viewmentioning

confidence: 99%

High–throughput automated platform for nuclear magnetic resonance–based structural proteomics

Vinarov

Markley

2005

Expert Review of Proteomics

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The development of new systems and strategies capable of synthesizing any desired soluble, labeled protein or protein fragment on a preparative scale is one of the most important tasks in biotechnology today. The Center for Eukaryotic Structural Genomics (WI, USA), in co-operation with Ehime University (Matsuyama, Japan) and CellFree Sciences Co., Ltd, has developed an automated platform for nuclear magnetic resonance-based structural proteomics that employs wheat germ extracts for cell-free production of labeled protein. The platform utilizes a single construct for all targets without any redesign of the DNA or RNA. Therefore, it offers advantages over commercial cell-free methods utilizing Escherichia coli extracts that require multiple constructs or redesign of the open reading frame. The protein production and labeling protocol is no more costly than E. coli cell-based approaches, is robust and scalable for high-throughput applications. This protocol has been used in the authors center to screen eukaryotic open reading frames from the Arabidopsis thaliana and human genomes and for the determination of nuclear magnetic resonance structures. With the recent addition of the GeneDecoder 1000 (CellFree Sciences Co., Ltd) robotic system, the Center for Eukaryotic Structural Genomics is able to carry out as many as 384 small-scale (50 microl) screening reactions per week. Furthermore, the Protemist (CellFree Sciences Co., Ltd) robotic system enables the Center for Eukaryotic Structural Genomics to carry out 16 production-scale (4 ml) reactions per week. Utilization of this automated platform technology to screen targets for expression and solubility and to produce stable isotope-labeled samples for nuclear magnetic resonance structure determinations is discussed.

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Dictyostelium discoideum as Expression Host: Isotopic Labeling of a Recombinant Glycoprotein for NMR Studies

Cited by 21 publications

References 25 publications

NMR assignment of prespore specific antigen—a cell surface adhesion glycoprotein from Dictyostelium discoideum

NMR assignment of prespore specific antigen—a cell surface adhesion glycoprotein from Dictyostelium discoideum

Efficient expression and primary purification of 6-his tagged human Fas ligand in Dictyostelium discoideum

High–throughput automated platform for nuclear magnetic resonance–based structural proteomics

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