Frits A de Wolf

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Recombinant non-hydroxylated gelatins based on mouse type I and rat type III collagen sequences were secreted from the methylotrophic yeast Pichia pastoris, using the Saccharomyces cerevisiae alpha-mating factor prepro signal. Proteolytic degradation could be minimized to a large extent by performing fermentations at pH 3.0 and by adding casamino acids to(More)
Heterologous proteins secreted by yeast and fungal expression hosts are occasionally degraded at basic amino acids. We cloned Pichia pastoris homologs of the Saccharomyces cerevisiae basic residue-specific endoproteases Kex2 and Yps1 to evaluate their involvement in the degradation of a secreted mammalian gelatin. Disruption of the P. pastoris KEX2 gene(More)
Several yeast systems have recently been developed for the recombinant production of gelatin and collagen. Amino acid sequence-specific prolyl 4-hydroxylation is essential for the gel-forming capacity of gelatin and for the proper folding of (pro)collagen. This post-translational modification is generally considered to be absent in microbial eukaryotic(More)
We report on self-healing, pH-responsive hydrogels that are entirely protein-based. The protein is a denovo designed recombinant triblock polypeptide of 66 kg/mol consisting of a silk-like middle block (GAGAGAGH)48, flanked by two long collagen-inspired hydrophilic random coil side blocks. The pH-dependent charge on the histidines in the silk block controls(More)
We have characterized the interaction of the antitumor drug doxorubicin with model membranes of the anionic phospholipids dioleoylphosphatidic acid (DOPA), dioleoylphosphatidylserine (DOPS), cardiolipin and dioleoylphosphatidylglycerol (DOPG) as compared to the zwitterionic dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylethanolamine (DOPE). The(More)
N-Hydroxysuccinimide (NHS)-ester-terminated monolayers were covalently attached in one step onto silicon using visible light. This mild photochemical attachment, starting from omega-NHS-functionalized 1-alkenes, yields a clean and flat monolayer-modified silicon surface and allows a mild and rapid functionalization of the surface by substitution of the(More)
We describe a straightforward approach to the covalent immobilization of cytophilic proteins by microcontact printing, which can be used to pattern cells on substrates. Cytophilic proteins are printed in micropatterns on reactive self-assembled monolayers by using imine chemistry. An aldehyde-terminated monolayer on glass or on gold was obtained by the(More)
A custom-designed, highly hydrophilic gelatin was produced in Pichia pastoris. Secreted production levels in single-copy transformants were in the range 3-6 g/l of clarified broth and purification to near homogeneity could be accomplished by differential ammonium sulfate precipitation. Despite the fact that gelatins are highly susceptible to proteolysis(More)
Anthracycline-membrane interactions play a role in the transport, the cytoplasmic distribution, and possibly also the activity of anthracyclines. Previous work on model membranes has shown that the widely-applied anticancer drug doxorubicin interacts specifically with anionic phospholipids [de Wolf, F. A., et al. (1991) Biochim. Biophys. Acta 106, 67-80].(More)
OmpF-Lpp, a model secretory protein, requires both a positively charged signal sequence and phosphatidylglycerol (PG) for efficient translocation across the E. coli inner membrane. Modification of the signal sequence can, however, remove both these prerequisites for translocation providing OmpF-Lpp mutants which undergo either PG and charge dependent or PG(More)