Expression of recombinant matrix components using baculoviruses

Mosher, Deane F.; Huwiler, Kristin G.; Misenheimer, Tina M.; Annis, Douglas S.

doi:10.1016/s0091-679x(02)69008-9

Cited by 40 publications

(57 citation statements)

References 14 publications

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“…FN was purified from a fibrinogen-rich plasma fraction by heat precipitation (60°C, 5 min) of the fibrinogen followed by chromatography (26). Expression and purification of polyhistidine-tagged monomeric [1][2][3][4][5] FNI, N-3 FNIII, [6][7][8][9] FNI, [7][8][9] FNI, 7 FNI-1 FNIII, [1][2][3][4][5][6][7][8][9][10][11][12][13][14] FNIII, and 2-14 FNIII and dimeric 6 FNI-C, 1 FNIII-C, and 2 FNIII-C were accomplished using recombinant baculovirus and affinity chromatography as described previously (27)(28)(29). Expression and purification of unlabeled [2][3] FNI and 8 -9 FNI, and of uniformly 15 N-labeled 8 -9 FNI, were performed as described previously (13,17).…”

Section: Methodsmentioning

confidence: 99%

Borrelia burgdorferi Protein BBK32 Binds to Soluble Fibronectin via the N-terminal 70-kDa Region, Causing Fibronectin to Undergo Conformational Extension

Harris

Maurer

et al. 2014

Journal of Biological Chemistry

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Background: The BBK32 adhesin of Borrelia burgdorferi interacts with fibronectin and contributes to infectivity. Results: BBK32 binds to fibronectin modules 2-5 FNI and 8 FNI through a tandem ␤-zipper and induces conformational change. Conclusion: The same extended site on fibronectin is targeted by different bacterial adhesins. Significance: Studies of the mechanism of BBK32-FN interaction enhance understanding of B. burgdorferi infection.

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Section: Methodsmentioning

confidence: 99%

Borrelia burgdorferi Protein BBK32 Binds to Soluble Fibronectin via the N-terminal 70-kDa Region, Causing Fibronectin to Undergo Conformational Extension

Harris

Maurer

et al. 2014

Journal of Biological Chemistry

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“…This product was cloned into the Xma1 and Pst1 sites of the baculovirus transfer vector pAcGP67.coco. The construct has an addition of 4 residues (ADPG) encoded by vector sequence N-terminal to the start of the FN gene and a short linker (SSAG) between the end of the 70K sequence (QTYP) and the six histidine residue tag (Mosher et al, 2002). r70K protein was expressed and purified using the His-tag as previously described (Mosher et al, 2002).…”

Section: Methodsmentioning

confidence: 99%

The N-terminal 70-kDa fragment of fibronectin binds to cell surface fibronectin assembly sites in the absence of intact fibronectin

2006

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Binding of the N-terminal 70-kDa (70K) fragment of fibronectin to fibroblasts blocks assembly of intact fibronectin and is an accurate indicator of the ability of various agents to enhance or inhibit fibronectin assembly. Such binding is widely thought to be to already assembled fibronectin. We evaluated this hypothesis with fibronectin-null mouse fibroblasts plated on laminin-1 in the absence of intact fibronectin. As a proteolytic fragment or recombinant protein, 70K bound fibronectin-null cells specifically in linear arrays that extended outwards from the periphery of spread cells. At early time points, these arrays were similar to those formed by intact fibronectin. 70K arrays formed within 5min following ligand addition at concentrations as low as 5nM, indicating rapid and high affinity binding. Bound 70K was extractable with Triton X-100 or deoxycholate but became insoluble when cross-linked with a membrane-impermeable agent into large SDS-stable complexes. Intact fibronectin, in contrast, became progressively non-extractable in the absence of cross-linking. The detergent-resistant arrays of cross-linked 70K localized to tips of cellular extensions and partially overlapped with α 6 and β 1 integrin subunits at the base of the extensions. α 5 did not localize with 70K arrays, but became progressively co-localized with assemblies of intact fibronectin over time. These results support a model in which the 70-kDa region of fibronectin binds to linearly arrayed cell surface molecules of adherent cells to initiate assembly, display of the arrays is controlled by the integrin that mediates adhesion, and fibronectin-binding integrins promote fibronectinfibronectin interactions during progression of assembly.

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“…The PCR products encoding 6d-VCAM-1 and 7d-VCAM-1 were 1771 and 2041 bp, respectively, and contained an XmaI site at the 5Ј end and an SpeI site at the 3Ј end. PCR fragments were digested with XmaI and SpeI restriction enzymes and cloned in-frame into the pAcGP67.coco plasmid (pCOCO) (28). The pCOCO plasmid containing the DNA insert encoding extracellular 7d-VCAM-1 was used as a template to generate the 4 -7VCAM-1, 1-3VCAM-1, and 1-2VCAM-1 constructs (Fig.…”

Section: Methodsmentioning

confidence: 99%

Differential Engagement of Modules 1 and 4 of Vascular Cell Adhesion Molecule-1 (CD106) by Integrins α4β1 (CD49d/29) and αMβ2 (CD11b/18) of Eosinophils

Barthel

Annis

Mosher

et al. 2006

Journal of Biological Chemistry

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We have studied adhesion of eosinophils to various forms of vascular cell adhesion molecule 1 (VCAM-1, CD106), an integrin counter-receptor implicated in eosinophil recruitment to the airway in asthma. Full-length 7d-VCAM-1, with seven immunoglobulin-like modules, contains integrin-binding sites in modules 1 and 4. The alternatively spliced six-module protein, 6d-VCAM-1, lacks module 4. In static assays, unactivated purified human blood eosinophils adhered similarly to recombinant soluble human 6d-VCAM-1 and 7d-VCAM-1 coated onto polystyrene microtiter wells. Further experiments, however, revealed differences in recognition of modules 1 and 4. Antibody blocking indicated that eosinophil adhesion to 6d-VCAM-1 or a VCAM-1 construct containing only modules 1-3, 1-3VCAM-1, is mediated by ␣4␤1 (CD49d/29) ,whereas adhesion to a construct containing modules 4 -7, 4 -7VCAM-1, is mediated by both ␣4␤1 and ␣M␤2 (CD11b/18). Inhibitors of phosphoinositide 3-kinase, which block adhesion of eosinophils mediated by ␣M␤2, blocked adhesion to 4 -7VCAM-1 but had no effect on adhesion to 6d-VCAM-1. Consistent with the antibody and pharmacological blocking experiments, eosinophilic leukemic cell lines lacking ␣M␤2 did not adhere to 4 -7VCAM-1 but did adhere to 6d-VCAM-1 or 1-3VCAM-1. Activation of eosinophils by interleukin (IL)-5 enhanced static adhesion to 6d-VCAM-1, 7d-VCAM-1, or 4 -7VCAM-1; IL-5-enhanced adhesion to all 3 constructs was blocked by anti-␣M␤2. Adhesion of unstimulated eosinophils to 7d-VCAM-1 under flow conditions was inhibited by anti-␣4 or anti-␣M. IL-5 treatment decreased eosinophil adhesion to 7d-VCAM-1 under flow, and anti-␣M had the paradoxical effect of increasing adhesion. These results demonstrate that ␣M␤2 modulates ␣4␤1-mediated eosinophil adhesion to VCAM-1 under both static and flow conditions. Vascular cell adhesion molecule 1 (VCAM-1)2 is a multimodular cell-surface glycoprotein up-regulated on human endothelium in response to pro-inflammatory cytokines in the asthmatic lung (1) and has been implicated in recruitment of eosinophils (EOS) from blood to the airway in asthma (2). Heterodimeric integrin receptors interacting with VCAM-1 are important in the rolling, firm adhesion, and movement of EOS (3, 4). These events are regulated by cytokines, which may alter integrin adhesive activity (5, 6). EOS express at least three potential integrin receptors that may be involved in adhesion to VCAM-1: ␣4␤1 (7-9), ␣D␤2 (7), and ␣4␤7 (8 -11).Considerable information is available about features of human VCAM-1 that are important for adhesion. The loop between ␤ strands C and D in the first immunoglobulin (Ig)-like module is accessible on the protein surface and contains the core integrin-recognition sequence Ile 39 -Asp-Ser-Pro-Leu 43 (IDSPL) (11-15). A second IDSPL site is present in the CD loop in module 4 (12, 14). Based on topology of exons within the mammalian genome and the similarity in amino acid sequence, a three-module unit was likely duplicated to generate VCAM-1 modules 1-3 and 4 -6 (16, 17). Modul...

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Expression of recombinant matrix components using baculoviruses

Cited by 40 publications

References 14 publications

Borrelia burgdorferi Protein BBK32 Binds to Soluble Fibronectin via the N-terminal 70-kDa Region, Causing Fibronectin to Undergo Conformational Extension

Borrelia burgdorferi Protein BBK32 Binds to Soluble Fibronectin via the N-terminal 70-kDa Region, Causing Fibronectin to Undergo Conformational Extension

The N-terminal 70-kDa fragment of fibronectin binds to cell surface fibronectin assembly sites in the absence of intact fibronectin

Differential Engagement of Modules 1 and 4 of Vascular Cell Adhesion Molecule-1 (CD106) by Integrins α4β1 (CD49d/29) and αMβ2 (CD11b/18) of Eosinophils

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