A time- and cost-efficient system for high-level protein production in mammalian cells

Aricescu, A. Radu; Lu, Wenjing; Jones, E Y

doi:10.1107/s0907444906029799

Cited by 719 publications

(781 citation statements)

References 31 publications

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“…The Fab fragment of OD01 was produced using the Pierce Mouse IgG1 Fab and F(ab′)2 Preparation Kit (Thermo Fischer Scientific) following the manufacturer's protocol. The eOD01 Fab fragment heavy-and light-chain genes were synthetized by GeneArt (Life Technologies), and the codon optimized cDNAs were cloned into the pHLsec vector (39). A C-terminal His 6 -tag was included in the heavy chain construct and both chains were coexpressed [1:1 (wt/wt) ratio of Fab heavy to light chain expressing plasmids] in HEK293T cells and purified, as described above.…”

Section: Methodsmentioning

confidence: 99%

“…For crystallization, a construct of JUNV GP1 (D87−N232) was derived by high-throughput cloning into the pOPINTTGneo mammalian expression vector (38). Constructs of MACV GP1 (E87-F257), LCMV GP1 (M81-K256), and an additional construct of JUNV GP1 with an amino acid C-terminal truncation (D87-V231; to facilitate cloning) were cloned into the pHLsec vector (39) for ELISA experiments. Proteins were expressed in transiently transfected HEK 293T cells (ATCC CRL-1573) in the presence of the α-mannosidase inhibitor, kifunensine, as previously described (39).…”

Section: Methodsmentioning

confidence: 99%

“…Constructs of MACV GP1 (E87-F257), LCMV GP1 (M81-K256), and an additional construct of JUNV GP1 with an amino acid C-terminal truncation (D87-V231; to facilitate cloning) were cloned into the pHLsec vector (39) for ELISA experiments. Proteins were expressed in transiently transfected HEK 293T cells (ATCC CRL-1573) in the presence of the α-mannosidase inhibitor, kifunensine, as previously described (39). Cell supernatants were harvested 4 d after transfection, clarified, and diafiltrated against a buffer containing 10 mM Tris (pH 8.0) and 150 mM NaCl (ÄKTA Flux diafiltration system; GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

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Convergent immunological solutions to Argentine hemorrhagic fever virus neutralization

Zeltina

Krumm

Mehmet

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Transmission of hemorrhagic fever New World arenaviruses from their rodent reservoirs to human populations poses substantial public health and economic dangers. These zoonotic events are enabled by the specific interaction between the New World arenaviral attachment glycoprotein, GP1, and cell surface human transferrin receptor (hTfR1). Here, we present the structural basis for how a mouse-derived neutralizing antibody (nAb), OD01, disrupts this interaction by targeting the receptor-binding surface of the GP1 glycoprotein from Junín virus (JUNV), a hemorrhagic fever arenavirus endemic in central Argentina. Comparison of our structure with that of a previously reported nAb complex (JUNV GP1-GD01) reveals largely overlapping epitopes but highly distinct antibody-binding modes. Despite differences in GP1 recognition, we find that both antibodies present a key tyrosine residue, albeit on different chains, that inserts into a central pocket on JUNV GP1 and effectively mimics the contacts made by the host TfR1. These data provide a molecular-level description of how antibodies derived from different germline origins arrive at equivalent immunological solutions to virus neutralization. . These viruses are endemic to rodent populations in rural areas of Argentina, Bolivia, and Venezuela, respectively, and viral spillover into human populations can result in severe hemorrhagic fever (HF) (3). Novel pathogenic NW arenaviruses continue to be identified (4-6), underscoring a wide-scale need for effective vaccines and therapeutics.JUNV, the etiological agent of Argentine hemorrhagic fever (AHF), constitutes one of the most dangerous NW arenaviruses, putting an estimated 5 million people at risk (7,8). JUNV infection typically exhibits a rapid onset of disease (7-14 d) and high mortality rates (15-30%) (7, 9, 10). There are no internationally approved drugs for preventing or treating NW arenavirus HF. However, the successful development of a live, attenuated JUNV vaccine, Candid#1, has proven that AHF can be controlled (11). Similarly, virus-neutralizing immune plasma from convalescent individuals has been successfully used for the treatment of AHF (10, 12).The JUNV envelope surface is decorated by trimeric multifunctional glycoprotein complex (GPC) spikes. Each protomer in the trimer consists of: a myristoylated (13) stable signal peptide, an attachment subunit (GP1), and a transmembrane fusion subunit (GP2) (14-19). Host-cell entry of JUNV and other clade B arenaviruses is initiated by the interaction between the arenaviral GP1 and the host transferrin receptor (TfR1) (20). The GP1 subunit interacts with the apical domain of TfR1, distal from natural transferrin and hereditary hemochromatosis protein recognition sites (21, 22). A primary determinant of zoonotic spread of clade B arenaviruses is the ability of the GP1 to recognize the human TfR1 ortholog (20, 23).The JUNV GPC spike comprises the primary target for neutralizing immune responses (24) and three GPC-specific mousederived nAbs-GD01, OD01, and GB03-have shown prom...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Convergent immunological solutions to Argentine hemorrhagic fever virus neutralization

Zeltina

Krumm

Mehmet

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…SOD1 fALS mutants (with the exception of G85R) were obtained by site-directed mutagenesis on the WT gene inside the pTH34 vector. The mutated genes were subcloned in the pHLsec vector between EcoRI and XhoI restriction enzymes 53 . All the clones were verified by DNA sequencing.…”

Section: Methodsmentioning

confidence: 99%

In-cell NMR reveals potential precursor of toxic species from SOD1 fALS mutants

et al. 2014

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Mutations in the superoxide dismutase 1 (SOD1) gene are related to familial cases of amyotrophic lateral sclerosis (fALS). Here we exploit in-cell NMR to characterize the protein folding and maturation of a series of fALS-linked SOD1 mutants in human cells and to obtain insight into their behaviour in the cellular context, at the molecular level. The effect of various mutations on SOD1 maturation are investigated by changing the availability of metal ions in the cells, and by coexpressing the copper chaperone for SOD1, hCCS. We observe for most of the mutants the occurrence of an unstructured SOD1 species, unable to bind zinc. This species may be a common precursor of potentially toxic oligomeric species, that are associated with fALS. Coexpression of hCCS in the presence of copper restores the correct maturation of the SOD1 mutants and prevents the formation of the unstructured species, confirming that hCCS also acts as a molecular chaperone.

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“…His 6 -tagged full-length prosaposin was expressed HEK293T cells using the pHLSec vector and purified as described previously (70).…”

Section: Methodsmentioning

confidence: 99%

Saposins modulate human invariant Natural Killer T cells self-reactivity and facilitate lipid exchange with CD1d molecules during antigen presentation

Salio

Ghadbane

Dushek³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Lipid transfer proteins, such as molecules of the saposin family, facilitate extraction of lipids from biological membranes for their loading onto CD1d molecules. Although it has been shown that prosaposin-deficient mice fail to positively select invariant natural killer T (iNKT) cells, it remains unclear whether saposins can facilitate loading of endogenous iNKT cell agonists in the periphery during inflammatory responses. In addition, it is unclear whether saposins, in addition to loading, also promote dissociation of lipids bound to CD1d molecules. To address these questions, we used a combination of cellular assays and demonstrated that saposins influence CD1d-restricted presentation to human iNKT cells not only of exogenous lipids but also of endogenous ligands, such as the self-glycosphingolipid β-glucopyranosylceramide, up-regulated by antigen-presenting cells following bacterial infection. Furthermore, we demonstrated that in human myeloid cells CD1d-loading of endogenous lipids after bacterial infection, but not at steady state, requires trafficking of CD1d molecules through an endo-lysosomal compartment. Finally, using BIAcore assays we demonstrated that lipid-loaded saposin B increases the offrate of lipids bound to CD1d molecules, providing important insights into the mechanisms by which it acts as a "lipid editor," capable of fine-tuning loading and unloading of CD1d molecules. These results have important implications in understanding how to optimize lipid-loading onto antigen-presenting cells, to better harness iNKT cells central role at the interface between innate and adaptive immunity. autoreactivity | inflammation | innate immunity | lipid binding proteins

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A time- and cost-efficient system for high-level protein production in mammalian cells

Cited by 719 publications

References 31 publications

Convergent immunological solutions to Argentine hemorrhagic fever virus neutralization

Convergent immunological solutions to Argentine hemorrhagic fever virus neutralization

In-cell NMR reveals potential precursor of toxic species from SOD1 fALS mutants

Saposins modulate human invariant Natural Killer T cells self-reactivity and facilitate lipid exchange with CD1d molecules during antigen presentation

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