Engineering hydrophobin DewA to generate surfaces that enhance adhesion of human but not bacterial cells

Boeuf, Stéphane; Throm, Tanja; Gutt, Beatrice; Strunk, Timo; Hoffmann, Marc P.; Seebach, Elisabeth; Mühlberg, Leonie; Brocher, Jan; Gotterbarm, Tobias; Wenzel, Wolfgang; Fischer, Reinhard; Richter, Wiltrud

doi:10.1016/j.actbio.2011.11.022

Cited by 34 publications

(32 citation statements)

References 34 publications

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“…Two class I hydrophobins from A. nidulans were chosen for fusion with the LccC laccase. The formation of monolayers on surfaces with the DewA protein is well characterized, and the protein has been previously modified with peptides without impairment of its coating ability (13,15). Another hydrophobin from A. nidulans, DewB, has the typical class I structure but also possesses a glycosylphosphatidylinositol (GPI) anchor for immobilization on the spore surface, where it contributes to its hydrophobicity (14).…”

Section: Resultsmentioning

confidence: 99%

“…Hydrophobin DewA with N-terminal or C-terminal peptide fusions have previously been used to coat wells of 96-well microtiter plates (15). Since fusion to a large enzyme, like laccase, could influence the binding ability of hydrophobins, different coating conditions and surfaces were tested.…”

Section: Resultsmentioning

confidence: 99%

“…To compare the immobilization on glass surfaces with different characteristics, cover glasses were also treated with Sigmacote (Sigma-Aldrich, Taufkirchen, Germany) to generate a hydrophobic surface prior to fusion protein immobilization. The coating procedure was adapted from previous studies (15). Filtered crude culture supernatants were diluted to 0.1 U/ml laccase activity (approximately 0.1 mg/ml total protein), directly applied on glass or polystyrene surfaces, and incubated overnight at 37°C in 50 mM sodium acetate buffer (pH 5) or sodium phosphate buffer (pH 7) with 1 mM CaCl 2 .…”

Section: Methodsmentioning

confidence: 99%

“…The filamentous fungus Aspergillus nidulans produces several class I hydrophobins, including a well-studied protein, DewA, which already showed great industrial potential and appears in several patent applications (13,14). DewA has been used as an emulsion stabilizer, in optimization of biliary stents, in the production of microcapsules, and was even fused to peptides for selective enhancement of human cell adhesion to surfaces (15)(16)(17). A DewA-enzyme fusion presents a tempting alternative to conventional methods of highly stable surface functionalization with an enzyme.…”

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confidence: 99%

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Immobilization of LccC Laccase from Aspergillus nidulans on Hard Surfaces via Fungal Hydrophobins

Fokina

Fenchel

Winandy

et al. 2016

Appl Environ Microbiol

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Fungal hydrophobins are small amphiphilic proteins that can be used for coatings on hydrophilic and hydrophobic surfaces. Through the formation of monolayers, they change the hydrophobicity of a given surface. Especially, the class I hydrophobins are interesting for biotechnology, because their layers are stable at high temperatures and can only be removed with strong solvents. These proteins self-assemble into monolayers under physiological conditions and undergo conformational changes that stabilize the layer structure. Several studies have demonstrated how the fusion of hydrophobins with short peptides allows the specific modification of the properties of a given surface or have increased the protein production levels through controlled localization of hydrophobin molecules inside the cell. Here, we fused the Aspergillus nidulans laccase LccC to the class I hydrophobins DewA and DewB and used the fusion proteins to functionalize surfaces with immobilized enzymes. In contrast to previous studies with enzymes fused to class II hydrophobins, the DewA-LccC fusion protein is secreted into the culture medium. The crude culture supernatant was directly used for coatings of glass and polystyrene without additional purification steps. The highest laccase surface activity was achieved after protein immobilization on modified hydrophilic polystyrene at pH 7. This study presents an easy-to-use alternative to classical enzyme immobilization techniques and can be applied not only for laccases but also for other biotechnologically relevant enzymes. IMPORTANCEAlthough fusion with small peptides to modify hydrophobin properties has already been performed in several studies, fusion with an enzyme presents a more challenging task. Both protein partners need to remain in active form so that the hydrophobins can interact with one another and form layers, and so the enzyme (e.g., laccase) will remain active at the same time. Also, because of the amphiphilic nature of hydrophobins, their production and purification remain challenging so far and often include steps that would irreversibly disrupt most enzymes. In our study, we present the first functional fusion proteins of class I hydrophobins from A. nidulans with a laccase. The resulting fusion enzyme is directly secreted into the culture medium by the fungus and can be used for the functionalization of hard surfaces. Immobilization of enzymes is of increasing importance in biotechnology. It provides various advantages compared to the application of free enzymes in solution, like increased stability, easy recovery, and reuse of the enzymes (1, 2). In some cases, binding to certain surfaces even improved enzyme activity (3). Several methods of immobilization are distinguished and are based on chemical and physical interactions between the enzyme and the surface, each having its own advantages and disadvantages (1, 2). Basic parameters, like maintenance of high enzyme activity, prevention of enzyme leaching, and contamination of the product, are relevant for choosing the ri...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Immobilization of LccC Laccase from Aspergillus nidulans on Hard Surfaces via Fungal Hydrophobins

Fokina

Fenchel

Winandy

et al. 2016

Appl Environ Microbiol

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“…Coating of plastic stents with hydrophobin led to a reduction in the amount of adsorbed material from human bile. DewA and fusion protein derivatives of this hydrophobin with Arg-Gly-Asp (RGD) or laminin globular domain (LG3) binding motif show increased adhesion of human cells (Boeuf et al 2012). In contrast, fusion of RGD and LG3 to DewA did not increase Staphylococcus aureus adhesion to the surfaces.…”

Section: Antifoulingmentioning

confidence: 99%

Applications of hydrophobins: current state and perspectives

Wösten

Scholtmeijer

2015

Appl Microbiol Biotechnol

142

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Hydrophobins are proteins exclusively produced by filamentous fungi. They self-assemble at hydrophilichydrophobic interfaces into an amphipathic film. This protein film renders hydrophobic surfaces of gas bubbles, liquids, or solid materials wettable, while hydrophilic surfaces can be turned hydrophobic. These properties, among others, make hydrophobins of interest for medical and technical applications. For instance, hydrophobins can be used to disperse hydrophobic materials; to stabilize foam in food products; and to immobilize enzymes, peptides, antibodies, cells, and anorganic molecules on surfaces. At the same time, they may be used to prevent binding of molecules. Furthermore, hydrophobins have therapeutic value as immunomodulators and can been used to produce recombinant proteins.

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Two forms and two faces, multiple states and multiple uses: Properties and applications of the self‐assembling fungal hydrophobins

2013

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The fungal hydrophobins are small proteins that are able to spontaneously self-assemble into amphipathic monolayers at hydrophobic:hydrophilic interfaces. These protein monolayers can reverse the wettability of a surface, making them suitable for increasing the biocompatibility of many hydrophobic materials. The self-assembling properties and amphipathic nature of hydrophobins make them attractive candidates for biotechnological applications. Recently, there have been significant advances in the understanding of the structure and assembly of these remarkable proteins. This opens up the way for engineering these proteins to encompass novel functions and for the use of hydrophobins in modification of nanomaterials. This review highlights the important structural aspects of the hydrophobins and the mechanisms by which they assemble and describes recent exciting developments in the use of hydrophobins for cell attachment, drug delivery, and protein purification.

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Engineering hydrophobin DewA to generate surfaces that enhance adhesion of human but not bacterial cells

Cited by 34 publications

References 34 publications

Immobilization of LccC Laccase from Aspergillus nidulans on Hard Surfaces via Fungal Hydrophobins

Immobilization of LccC Laccase from Aspergillus nidulans on Hard Surfaces via Fungal Hydrophobins

Applications of hydrophobins: current state and perspectives

Two forms and two faces, multiple states and multiple uses: Properties and applications of the self‐assembling fungal hydrophobins

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