Expression, production, and characterization of full-length vitronectin in Escherichia coli

Wojciechowski, Katherine; Chang, Chien-Hsing; Hocking, Denise C.

doi:10.1016/j.pep.2004.04.004

Cited by 16 publications

(14 citation statements)

References 21 publications

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“…Thus, cell proliferation assays were used in the present study to assess the functional state of fibronectin fibrils. To examine the effect of substrate adhesion on the proliferative response to ECM fibronectin, FN-null MEFs were seeded onto wells pre-coated with saturating concentrations of either collagen or vitronectin (Sottile et al, 1998; Wojciechowski et al, 2004), in the absence or presence of increasing concentrations of fibronectin. Cell number was determined after 4 days (Sottile et al, 1998).…”

Section: Resultsmentioning

confidence: 99%

“…The following fusion proteins were produced in bacteria and isolated by affinity chromatography as described previously: GST-tagged vitronectin (Wojciechowski et al, 2004), GST/III1H,8-10 (Hocking and Kowalski, 2002), GST/III1H,8-10ΔRWRK (Roy et al, 2010), polyhistidine (His)-tagged III1C (Morla et al, 1994), His-III11C (Morla et al, 1994). The molecular mass of III1C is similar to that of the control, III11C (Morla et al, 1994).…”

Section: Methodsmentioning

confidence: 99%

“…Fibronectin-null mouse embryonic fibroblasts (FN-null MEFs), cultured in defined serum- and fibronectin-free growth media, were used to directly compare the mechanisms and cellular effects of vitronectin-mediated fibronectin matrix assembly with that of collagen-mediated assembly. FN-null MEFs polymerize exogenously-added fibronectin into ECM fibrils via a process indistinguishable from that of fibronectin-expressing cells (Sottile et al, 1998), and do not express detectable levels of vitronectin (Wojciechowski et al, 2004). Thus, this model permits tight control over the composition of the adhesive substrate, the amount of fibronectin to which the cells are exposed, and the initiation and timing of the matrix assembly process.…”

Section: Introductionmentioning

confidence: 99%

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Opposing effects of collagen I and vitronectin on fibronectin fibril structure and function

et al. 2014

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Extracellular matrix fibronectin fibrils serve as passive structural supports for the organization of cells into tissues, yet can also actively stimulate a variety of cell and tissue functions, including cell proliferation. Factors that control and coordinate the functional activities of fibronectin fibrils are not known. Here, we compared effects of cell adhesion to vitronectin versus type I collagen on the assembly of and response to, extracellular matrix fibronectin fibrils. The amount of insoluble fibronectin matrix fibrils assembled by fibronectin-null mouse embryonic fibroblasts adherent to collagen- or vitronectin-coated substrates was not significantly different 20 h after fibronectin addition. However, the fibronectin matrix produced by vitronectin-adherent cells was ~10-fold less effective at enhancing cell proliferation than that of collagen-adherent cells. Increasing insoluble fibronectin levels with the fibronectin fragment, anastellin did not increase cell proliferation. Rather, native fibronectin fibrils polymerized by collagen- and vitronectin-adherent cells exhibited conformational differences in the growth-promoting, III-1 region of fibronectin, with collagen-adherent cells producing fibronectin fibrils in a more extended conformation. Fibronectin matrix assembly on either substrate was mediated by α5β1 integrins. However, on vitronectin-adherent cells, α5β1 integrins functioned in a lower activation state, characterized by reduced 9EG7 binding and decreased talin association. The inhibitory effect of vitronectin on fibronectin-mediated cell proliferation was localized to the cell-binding domain, but was not a general property of αvβ3 integrin-binding substrates. These data suggest that adhesion to vitronectin allows for the uncoupling of fibronectin fibril formation from downstream signaling events by reducing α5β1 integrin activation and fibronectin fibril extension.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Opposing effects of collagen I and vitronectin on fibronectin fibril structure and function

et al. 2014

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“…Many obstacles are encountered, however, when using these fusion partners. In some cases the fusion partner cannot be efficiently removed from the target proteins and this could interfere with downstream applications (Wojciechowski et al, 2004). In addition, many target proteins have been observed to precipitate after removal of the fusion partner (Donnelly et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

EspA is a novel fusion partner for expression of foreign proteins in Escherichia coli

Cheng

Wang

et al. 2010

Journal of Biotechnology

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“…Wojciechowski et al (3) were the first to report expression of rhVTN in E. coli , and these authors described its purification based on fusion to a glutathione S-transferase (GST) tag. Chen et al (4), in establishing chemically defined culture conditions for human embryonic stem (ES) and induced pluripotent stem cells, modified the procedure to facilitate purification of bioactive untagged rhVTNand truncated derivatives thereofby exploiting VTN's heparin binding activity in 8 M urea.…”

mentioning

confidence: 99%

Expression and purification of bioactive, low-endotoxin recombinant human vitronectin

Halford

Stacker

2014

BioTechniques

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The secreted adhesive glycoprotein vitronectin (VTN) is a multifunctional component of plasma and the extracellular matrix. A high-yielding, inexpensive, low endotoxin source of bioactive recombinant human vitronectin (rhVTN) is highly desirable for in vitro use in diverse cell culture systems ranging from basic research settings to clinical-grade production of human cells. We describe modifications to a previously reported heparin-based affinity chromatography procedure that improve yield and achieve efficient removal of endotoxin from washed and urea-solubilized human VTN inclusion bodies following standard autoinduction of expression in Escherichia coli. This simple procedure makes accessible the low-cost expression and purification of large quantities of bioactive rhVTN using basic equipment and facilitates its use in a spectrum of endotoxin-sensitive applications.

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Expression, production, and characterization of full-length vitronectin in Escherichia coli

Cited by 16 publications

References 21 publications

Opposing effects of collagen I and vitronectin on fibronectin fibril structure and function

Opposing effects of collagen I and vitronectin on fibronectin fibril structure and function

EspA is a novel fusion partner for expression of foreign proteins in Escherichia coli

Expression and purification of bioactive, low-endotoxin recombinant human vitronectin

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