Rapid expression of an anti-human C5 chimeric Fab utilizing a vector that replicates in COS and 293 cells

Evans, Mark J.; Hartman, Sandra L.; Wolff, Dennis W.; Rollins, Scott A.; Squinto, Stephen P.

doi:10.1016/0022-1759(95)00093-p

Cited by 42 publications

(26 citation statements)

References 35 publications

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“…Coding regions were amplified by PCR and incorporated into expression vectors based on pApex-3 (28). The cloning strategies used resulted in expressed proteins that were tagged at the N terminus with either the Flag octapeptide or with GST.…”

Section: Methodsmentioning

confidence: 99%

Poxvirus ankyrin repeat proteins are a unique class of F-box proteins that associate with cellular SCF1 ubiquitin ligase complexes

Sonnberg

Seet

Pawson

et al. 2008

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

120

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F-box proteins direct the degradation of an extensive range of proteins via the ubiquitin-proteasome system. Members of this large family of proteins are typically bipartite. They recruit specific substrates through a substrate-binding domain and, via the F-box, link these to core components of a major class of ubiquitin ligases (SCF1). F-box proteins thus determine the specificity of SCF1-mediated ubiquitination. F-box-like motifs were recently detected in poxvirus ankyrin repeat (ANK) proteins but clear compositional differences to typical F-box proteins raise questions regarding the classification and function of the motif. Here we show that all five ANK proteins of a representative poxvirus, Orf virus, interact in vivo with core components of the SCF1 ubiquitin ligase complex. Interaction is dependent on the poxviral F-box-like motif and the adaptor subunit of the complex (SKP1). The viral protein does not block enzymatic activity of the complex. These observations identify the poxviral motif as a functional F-box. They also identify a new class of F-box that in contrast to cellular counterparts is truncated, has an extreme C-terminal location and is paired with an ANK protein-binding domain. ANK proteins constitute the largest family of poxviral proteins but their function and the significance of their abundance have remained an enigma. We propose that poxviruses use these unique ANK/F-box proteins to dictate target specificity to SCF1 ubiquitin ligases and thereby exploit the cell's ubiquitin-proteasome machinery.

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Section: Methodsmentioning

confidence: 99%

Poxvirus ankyrin repeat proteins are a unique class of F-box proteins that associate with cellular SCF1 ubiquitin ligase complexes

Sonnberg

Seet

Pawson

et al. 2008

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

120

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“…A construct encoding a heavy-chain leader sequence (TIB142; American Type Culture Collection, Manassas, VA) and an IgA-Fc region (C242 to P455; IgA1 myeloma Bur numbering) from IMAGE clone 4701069 (GenBank accession no. BC016369) was expressed from pAPEX-3p-X-DEST (pBAR424), a Gateway RfA cassette (Invitrogen, Carlsbad, CA) derivative of pAPEX-3p (44). The IgA-Fc was produced by transfection of HEK293EBNA cells with Lipofectamine 2000 (Invitrogen) and selection with 2 g/ml puromycin (Sigma, Melbourne, Australia).…”

Section: Methodsmentioning

confidence: 99%

Structural basis for evasion of IgA immunity by Staphylococcus aureus revealed in the complex of SSL7 with Fc of human IgA1

Ramsland

Willoughby

Trist

et al. 2007

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

106

102

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Infection by Staphylococcus aureus can result in severe conditions such as septicemia, toxic shock, pneumonia, and endocarditis with antibiotic resistance and persistent nasal carriage in normal individuals being key drivers of the medical impact of this virulent pathogen. In both virulent infection and nasal colonization, S. aureus encounters the host immune system and produces a wide array of factors that frustrate host immunity. One in particular, the prototypical staphylococcal superantigen-like protein SSL7, potently binds IgA and C5, thereby inhibiting immune responses dependent on these major immune mediators. We report here the three-dimensional structure of the complex of SSL7 with Fc of human IgA1 at 3.2 Å resolution. Two SSL7 molecules interact with the Fc (one per heavy chain) primarily at the junction between the C␣2 and C␣3 domains. The binding site on each IgA chain is extensive, with SSL7 shielding most of the lateral surface of the C␣3 domain. However, the SSL7 molecules are positioned such that they should allow binding to secretory IgA. The key IgA residues interacting with SSL7 are also bound by the leukocyte IgA receptor, Fc␣RI (CD89), thereby explaining how SSL7 potently inhibits IgAdependent cellular effector functions mediated by Fc␣RI, such as phagocytosis, degranulation, and respiratory burst. Thus, the ability of S. aureus to subvert IgA-mediated immunity is likely to facilitate survival in mucosal environments such as the nasal passage and may contribute to systemic infections.Staphylococcus aureus is an important human pathogen causing conditions ranging from minor superficial skin infections to life-threatening syndromes, including sepsis, toxic shock syndrome, osteomyelitis, pneumonitis, and endocarditis. It is carried without symptoms in at least 20% of individuals (1, 2). The emergence in the 1960s of pandemic penicillin-resistant S. aureus has been followed by a variety of hospital-associated and community-associated methicillin-resistant strains (HA-and CA-MRSA) (2, 3). The increased prevalence of MRSA infections and corresponding rise in life-threatening syndromes have made it imperative to elucidate the mechanisms of pathogenesis for S. aureus. The interaction between S. aureus and the host is complex and is mediated by an array of bacterial proteins that both mediate the various pathologies and modify the immune system of the host (4-10).SSL7 (formerly named SET1) is the first described member of a new family of putative S. aureus toxins, the staphylococcal superantigen-like (SSL) proteins (11, 12), related to the staphylococcal enterotoxins (SEs) or superantigens (13). The SSL proteins have Ϸ30% sequence identity with toxic shock syndrome 1 (TSST-1) and 25% or less identity with other SEs. Despite the sequence differences, the SSL proteins have a typical SE tertiary structure consisting of a distinct oligonucleotide/oligosaccharide binding (OB-fold) linked to a ␤-grasp domain (14 -16).Similar to the se genes, the ssl genes are located in a pathogenicity island (SaPIn2) and ...

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“…Also available were equivalent vectors, containing nucleotides 4 -401 from Orf virus strain NZ2 (21), nucleotides 4 -458 from Pseudocowpox virus strain VR634 (27), and nucleotides 4 -576 of mouse VEGF 164 (33). XbaI fragments from the vectors described above, containing the VEGF coding regions and FLAG octapeptide, were subcloned into the mammalian expression vector, pA-PEX-3 (34). Protein synthesis from these vectors would therefore give rise to secreted polypeptides that were tagged with the FLAG octapeptide at their C termini.…”

Section: Methodsmentioning

confidence: 99%

Viral Vascular Endothelial Growth Factors Vary Extensively in Amino Acid Sequence, Receptor-binding Specificities, and the Ability to Induce Vascular Permeability yet Are Uniformly Active Mitogens

Wise

Ueda

Dryden

et al. 2003

Journal of Biological Chemistry

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Infections of humans and ungulates by parapoxviruses result in skin lesions characterized by extensive vascular changes that have been linked to viral-encoded homologues of vascular endothelial growth factor (VEGF). VEGF acts via a family of receptors (VEGFRs) to mediate endothelial cell proliferation, vascular permeability, and angiogenesis. The VEGF genes from independent parapoxvirus isolates show an extraordinary degree of inter-strain sequence variation. We conducted functional comparisons of five representatives of the divergent viral VEGFs. These revealed that despite the sequence divergence, all were equally active mitogens, stimulating proliferation of human endothelial cells in vitro and vascularization of sheep skin in vivo with potencies equivalent to VEGF. This was achieved even though the viral VEGFs bound VEGFR-2 less avidly than did VEGF. Surprisingly the viral VEGFs varied in their ability to cross-link VEGFR-2, induce vascular permeability and bind neuropilin-1. Correlations between these three activities were detected. In addition it was possible to correlate these functional variations with certain sequence and structural motifs specific to the viral VEGFs. In contrast to the conserved ability to bind human VEGFR-2, the viral growth factors did not bind either VEGFR-1 or VEGFR-3. We propose that the extensive sequence divergence seen in the viral VEGFs was generated primarily by selection against VEGFR-1 binding.

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Rapid expression of an anti-human C5 chimeric Fab utilizing a vector that replicates in COS and 293 cells

Cited by 42 publications

References 35 publications

Poxvirus ankyrin repeat proteins are a unique class of F-box proteins that associate with cellular SCF1 ubiquitin ligase complexes

Poxvirus ankyrin repeat proteins are a unique class of F-box proteins that associate with cellular SCF1 ubiquitin ligase complexes

Structural basis for evasion of IgA immunity by Staphylococcus aureus revealed in the complex of SSL7 with Fc of human IgA1

Viral Vascular Endothelial Growth Factors Vary Extensively in Amino Acid Sequence, Receptor-binding Specificities, and the Ability to Induce Vascular Permeability yet Are Uniformly Active Mitogens

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