A multifunctional plasmid for protein expression by ECPCR: overproduction of the p50 subunit of NF-κB

Sodeoka, Mikiko; Larson, Christopher J.; Chen, Lin; LeClair, Kenneth; Verdine, Gregory L.

doi:10.1016/s0960-894x(00)80293-0

Cited by 51 publications

(40 citation statements)

References 26 publications

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“…AAQ73926) from genomic DNA of the A4 strain (also called FCR3 or IT4) of P. falciparum in the pLM1 expression plasmid (21). The plasmid was transformed into Escherichia coli strain BL21 (DE3)-RIL (Stratagene, La Jolla, CA) cells that were then grown to an A 600 of 0.6 at 37°C and induced with 1 mM isopropyl-␤-D-thiogalactopyranoside for 3 h to produce insoluble protein as inclusion bodies.…”

Section: Methodsmentioning

confidence: 99%

Subdomain 3 of Plasmodium falciparum VAR2CSA DBL3x Is Identified as a Minimal Chondroitin Sulfate A-binding Region

Singh

Gitti²,

Diouf

et al. 2010

Journal of Biological Chemistry

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Molecular interactions between the VAR2CSA protein, expressed on the surface of Plasmodium falciparum-infected erythrocytes, and placental chondroitin sulfate A (CSA) are primarily responsible for pregnancy-associated malaria (PAM). Interrupting these interactions may prevent or ameliorate the severity of PAM. Several of the Duffy binding-like (DBL) domains of VAR2CSA, including the DBL3x domain, have been shown to bind CSA in vitro, but a more detailed understanding of how DBL domains bind CSA is needed. In this study, we demonstrate that subdomain 3 (S3), one of the three subdomains of VAR2CSA DBL3x by itself, is the major contributor toward CSA binding. NMR spectroscopy and flow cytometry analyses show that S3 and the intact DBL3x domain bind CSA similarly. Mutations within the S3 portion of DBL3x markedly affect CSA binding. Both recombinant molecules, S3 and DBL3x, are recognized by antibodies in the plasma of previously pregnant women living in malaria-endemic regions of Mali, but much less so by plasma from men of the same regions. As the S3 sequence is highly conserved in all known VAR2CSA proteins expressed by different parasite isolates obtained from various malaria endemic areas of the world, the identification of S3 as an independent CSA-binding region provides a compelling molecular basis for designing interventions against PAM.Each year, pregnancy-associated malaria (PAM) 3 poses a risk to millions of women worldwide. Although women living in malaria-endemic areas eventually develop immunity to malaria after repeated exposure to Plasmodium falciparum, they become susceptible to severe disease during pregnancy. PAM is caused by the massive sequestration of P. falciparum-infected erythrocytes (IEs) that selectively bind to CSA in the placenta, leading to severe inflammation and life-threatening outcomes for both mother and child (1).VAR2CSA, a member of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family, is primarily responsible for the binding of IEs to CSA (2). The var2csa gene is found in nearly all P. falciparum isolates (3) and is unusually conserved compared with other members of the var gene family that encode PfEMP1 molecules (4). VAR2CSA is specifically up-regulated in infected erythrocytes that are either isolated from the placenta (5) or selected in vitro for binding to CSA (2). On disruption of the var2csa gene, the ability of the infected erythrocytes to adhere to CSA is lost (6) or greatly reduced (7).The risk of PAM is highest in the first pregnancy due to lack of VAR2CSA-specific antibodies, but with increasing parity, women acquire such antibodies that block the binding of IEs to CSA in the placenta. The naturally acquired antibodies from multiparous women collected from different malaria-endemic regions of the world recognize CSA-binding IEs in the placenta (8 -12). The ability of such antibodies to block adhesion of IEs to CSA by recognizing conserved epitopes among different parasite clones, suggests that VAR2CSA-based vaccination against PAM is possible.Expressing...

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Section: Methodsmentioning

confidence: 99%

Subdomain 3 of Plasmodium falciparum VAR2CSA DBL3x Is Identified as a Minimal Chondroitin Sulfate A-binding Region

Singh

Gitti²,

Diouf

et al. 2010

Journal of Biological Chemistry

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“…Human CD3-and CD3-␦ ectodomain constructs were each modified by PCR and inserted into a bacterial expression plasmid, pLM1 (29). The CD3-construct encodes residues 1-105, and the CD3-␦ construct encodes residues 1-79.…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of a human CD3-ε/δ dimer in complex with a UCHT1 single-chain antibody fragment

Arnett

Harrison

Wiley

2004

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

138

133

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The ␣͞␤ T cell receptor complex transmits signals from MHC͞ peptide antigens through a set of constitutively associated signaling molecules, including CD3-͞␥ and CD3-͞␦. We report the crystal structure at 1.9-Å resolution of a complex between a human CD3-͞␦ ectodomain heterodimer and a single-chain fragment of the UCHT1 antibody. CD3-͞␦ and CD3-͞␥ share a conserved interface between the Ig-fold ectodomains, with parallel packing of the two G strands. CD3-␦ has a more electronegative surface and a more compact Ig fold than CD3-␥; thus, the two CD3 heterodimers have distinctly different molecular surfaces. The UCHT1 antibody binds near an acidic region of CD3-opposite the dimer interface, occluding this region from direct interaction with the TCR. This immunodominant epitope may be a uniquely accessible surface in the TCR͞CD3 complex, because there is overlap between the binding site of the UCHT1 and OKT3 antibodies. Determination of the CD3-͞␦ structure completes the set of TCR͞CD3 globular ectodomains and contributes information about exposed CD3 surfaces.antibody-binding site ͉ CD3 ͉ T cell receptor T he ␣͞␤ T cell receptor (TCR) is a multimeric cell-surface complex comprising a clonotypic antigen-binding TCR heterodimer and three conserved signal transducing modules: CD3-͞␥ and CD3-͞␦ heterodimers and a TCR-homodimer (1-3). TCR͞CD3 chains assemble into a minimal eight-subunit complex in the endoplasmic reticulum through a series of dimeric and trimeric interactions with a stoichiometry of one TCR-␣͞␤ heterodimer, one CD3-͞␥ heterodimer, one CD3-͞␦ heterodimer, and a TCR-homodimer (4-8). Whereas extracellular contacts are sufficient for interactions within TCR and CD3 heterodimers (9-12), transmembrane interactions are necessary for assembly and surface expression of intact TCR͞CD3 complexes (7,(13)(14)(15)(16)(17)(18). Extracellular domains of CD3 may provide additional specificity to mandatory transmembrane interactions (8,19,20), but specific extracellular interactions between TCR and CD3-͞␦ or CD3-͞␥ have yet to be determined.Antibodies to CD3, first used to identify and characterize T cells, are effective laboratory tools for activation of many pathways in the T cell signaling cascade. One such antibody is UCHT1 (21). Fusions of UCHT1 with immunotoxins are being used for targeted T cell depletion in animal models of organ transplantation (22). UCHT1 and several other anti-CD3 antibodies, including OKT3, recognize epitopes on CD3-found in heterodimers with CD3-␦ and with CD3-␥, but not CD3-expressed in isolation (23). Understanding the specific interactions between UCHT1 and CD3 may facilitate development of this or other antibodies as clinical agents. Moreover, because UCHT1 immunoprecipitates all TCR and CD3 components, it is a good tool for studying accessible surfaces of CD3 in the TCR͞CD3 complex.Detailed structural knowledge of the TCR͞CD3 complex is important for understanding how the TCR transmits the signal it receives from its MHC͞peptide ligand. The structure of the TCR͞CD3 complex and its relati...

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“…Protein expression was induced by the addition of galactose to a final concentration of 2%. After 4 h, ␤-galactosidase activity was quantified as described (30) Recombinant Proteins-The recombinant NF-B p50 RHR (residues 1-366 or residues 1-366 with deletion of 2-39) was expressed and purified to homogeneity as described (33). The p65 RHR (residues 1-305) was the generous gift of R. Chopra and S. Harrison (Harvard University).…”

Section: Methodsmentioning

confidence: 99%

A Small Region in HMG I(Y) Is Critical for Cooperation with NF-κB on DNA

Zhang

Verdine

1999

Journal of Biological Chemistry

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A multifunctional plasmid for protein expression by ECPCR: overproduction of the p50 subunit of NF-κB

Cited by 51 publications

References 26 publications

Subdomain 3 of Plasmodium falciparum VAR2CSA DBL3x Is Identified as a Minimal Chondroitin Sulfate A-binding Region

Subdomain 3 of Plasmodium falciparum VAR2CSA DBL3x Is Identified as a Minimal Chondroitin Sulfate A-binding Region

Crystal structure of a human CD3-ε/δ dimer in complex with a UCHT1 single-chain antibody fragment

A Small Region in HMG I(Y) Is Critical for Cooperation with NF-κB on DNA

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