Biomedical subjects

Joseph G H Wessels

Publications and source records attributed to Joseph G H Wessels.

6 recordsLinked to original sources

PubMed2004

Highly efficient production of laccase by the basidiomycete Pycnoporus cinnabarinus.

An efficient transformation and expression system was developed for the industrially relevant basidiomycete Pycnoporus cinnabarinus. This was used to transform a laccase-deficient monokaryotic strain with the homologous lac1 laccase gene placed under the regulation of its own promoter or that of the SC3 hydrophobin gene or the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Schizophyllum commune. SC3-driven expression resulted in a maximal laccase activity of 107 nkat ml(-1) in liquid shaken cultures. This value was about 1.4 and 1.6 times higher in the cases of the GPD and lac1 promoters, respectively. lac1-driven expression strongly increased when 25 g of ethanol liter(-1) was added to the medium. Accordingly, laccase activity increased to 1,223 nkat ml(-1). These findings agree with the fact that ethanol induces laccase gene expression in some fungi. Remarkably, lac1 mRNA accumulation and laccase activity also strongly increased in the presence of 25 g of ethanol liter(-1) when lac1 was expressed behind the SC3 or GPD promoter. In the latter case, a maximal laccase activity of 1,393 nkat ml(-1) (i.e., 360 mg liter(-1)) was obtained. Laccase production was further increased in transformants expressing lac1 behind its own promoter or that of GPD by growth in the presence of 40 g of ethanol liter(-1). In this case, maximal activities were 3,900 and 4,660 nkat ml(-1), respectively, corresponding to 1 and 1.2 g of laccase per liter and thus representing the highest laccase activities reported for recombinant fungal strains. These results suggest that P. cinnabarinus may be a host of choice for the production of other proteins as well.

Basidiomycota↗

PubMed2002

Self-assembly of the hydrophobin SC3 proceeds via two structural intermediates.

Hydrophobins self assemble into amphipathic films at hydrophobic-hydrophilic interfaces. These proteins are involved in a broad range of processes in fungal development. We have studied the conformational changes that accompany the self-assembly of the hydrophobin SC3 with polarization-modulation infrared reflection absorption spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, and circular dichroism, and related them to changes in morphology as observed by electron microcopy. Three states of SC3 have been spectroscopically identified previously as follows: the monomeric state, the alpha-helical state that is formed upon binding to a hydrophobic solid, and the beta-sheet state, which is formed at the air-water interface. Here, we show that the formation of the beta-sheet state of SC3 proceeds via two intermediates. The first intermediate has an infrared spectrum indistinguishable from that of the alpha-helical state of SC3. The second intermediate is rich in beta-sheet structure and has a featureless appearance under the electron microscope. The end state has the same secondary structure, but is characterized by the familiar 10-nm-wide rodlets.

Bacterial Proteins↗

PubMed2002

Surface modifications created by using engineered hydrophobins.

Hydrophobins are small (ca. 100 amino acids) secreted fungal proteins that are characterized by the presence of eight conserved cysteine residues and by a typical hydropathy pattern. Class I hydrophobins self-assemble at hydrophilic-hydrophobic interfaces into highly insoluble amphipathic membranes, thereby changing the nature of surfaces. Hydrophobic surfaces become hydrophilic, while hydrophilic surfaces become hydrophobic. To see whether surface properties of assembled hydrophobins can be changed, 25 N-terminal residues of the mature SC3 hydrophobin were deleted (TrSC3). In addition, the cell-binding domain of fibronectin (RGD) was fused to the N terminus of mature SC3 (RGD-SC3) and TrSC3 (RGD-TrSC3). Self-assembly and surface activity were not affected by these modifications. However, physiochemical properties at the hydrophilic side of the assembled hydrophobin did change. This was demonstrated by a change in wettability and by enhanced growth of fibroblasts on Teflon-coated with RGD-SC3, TrSC3, or RGD-TrSC3 compared to bare Teflon or Teflon coated with SC3. Thus, engineered hydrophobins can be used to functionalize surfaces.

Amino Acid Sequence↗

PubMed1998

Identification of three differentially expressed hydrophobins in Pleurotus ostreatus (oyster mushroom).

Three proteins with characteristic features of class I hydrophobins, designated POH1, POH2 and POH3, were isolated from the basidiomycete Pleurotus ostreatus. Based on N-terminal sequence analyses, their cDNAs were isolated using RT-PCR; the cDNAs and corresponding genes were sequenced and their regulation studied. POH1 is expressed in the fruiting bodies but not in vegetative mycelium. The regulation of POH2 and POH3 is tightly correlated. Both genes are switched off in the fruiting bodies but abundantly expressed in the vegetative mycelium of both monokaryon and dikaryon. POH2 and POH3 were isolated from the culture medium and from aerial hyphae. Co-purified POH2 and POH3 assembled in vitro into a protein membrane with a typical rodlet pattern as found previously with other hydrophobins. Similar structures were detected on the surface of aerial hyphae.

Amino Acid Sequence↗

PubMed1998

A hydrophobin (ABH3) specifically secreted by vegetatively growing hyphae of Agaricus bisporus (common white button mushroom).

Aerial mycelium and hyphal strands of Agaricus bisporus, strain U1, exhibited a rodlet pattern at their surfaces characteristic for assembled class I hydrophobins. An SDS-insoluble/trifluoroacetic-acid-soluble fraction from strands was found to contain one abundant protein with an apparent molecular mass on gel of 19 kDa. Two sequences for this protein (ABH3), typical of class I hydrophobins, could be deduced by sequencing cDNA clones obtained by RT-PCR. The two forms of the protein could be assigned to different alleles present in the two homokaryons that constitute the heterokaryotic U1 strain. ABH3 displays all the in vitro properties of a typical class I hydrophobin such as SC3 from Schizophyllum commune but is not glycosylated or otherwise post-translationally modified because the molecular mass values deduced from the amino acid sequence (9228 and 9271 Da) and derived from mass spectrometry were in good agreement. The ABH3 transcript was found to be present in the vegetative mycelium of both primary and secondary mycelium but not in the fruiting bodies, whereas the reverse was found for the ABH1 hydrophobin. Using an S. commune mutant with a disrupted SC3 gene it was found that ABH3 can substitute for SC3 in inducing formation of aerial hyphae, suggesting a role of ABH3 in the emergence of aerial hyphae and strands in A. bisporus.

Agaricus↗

PubMed1996

An abundant hydrophobin (ABH1) forms hydrophobic rodlet layers in Agaricus bisporus fruiting bodies.

The SDS-insoluble protein fraction of Agaricus bisporus fruiting bodies was solubilized with trifluoroacetic acid. On SDS-PAGE this fraction was found to contain one abundant protein with an apparent M(r) of 16 kDa. The N-terminal amino acid sequence of this protein was determined and RT-PCR used to isolate a cDNA clone which upon sequencing identified the protein as a typical class I hydrophobin (ABH1). The gene (ABH1) was isolated and sequenced, and a second hydrophobin gene (ABH2) was found about 2.5 kbp downstream of ABH1. Purified ABH1 self-assembled at hydrophobic-hydrophilic interfaces, producing the typical rodlet layer known from other hydrophobins. Similar rodlets were observed on the surface of the fruiting body, while immunological localization showed the hydrophobin to be particularly abundant at the outer surface of fruiting bodies, in the veil and in the core tissue of the stipe. Transcripts of ABH1 were found only in fruiting-body hyphae. The ABH1 hydrophobin is probably solely responsible for the hydrophobicity of the fruiting-body surface but may also line air channels within fruiting bodies.

Agaricus↗