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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)
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)
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)
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)
Genetically engineered protein polymers (GEPP) are a class of multifunctional materials with precisely controlled molecular structure and property profile. Representing a promising alternative for currently used materials in biomedical applications, GEPP offer multiple benefits over natural and chemically synthesized polymers. However, producing them in(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)
Previously, we have shown that gel-forming triblock proteins, consisting of random coil middle blocks and trimer-forming (Pro-Gly-Pro)(9) end blocks, are efficiently produced and secreted by the yeast Pichia pastoris. These end blocks had a melting temperature (T(m)) of ∼41°C (at 1.1 mM of protein). The present work reveals that an increase of T(m) to(More)
Like natural tropoelastin, polypeptides based on an elastin-like VPGXG repeat have a characteristic inverse temperature response, which leads to coacervate formation above a certain transition temperature and which could be useful for a variety of applications. The key advantage of elastin-like polypeptides (ELPs) over (tropo)elastin is a full control over(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)
Microiontophoretic delivery of horseradish peroxidase in the torus semicircularis of the trout resulted in heavy labeling of somata in the rhombencephalic nucleus intermedius octavolateralis and nucleus octavus magnocellularis. In addition some labeled somata were found closely to the fasciculus longitudinalis lateralis and in the diencephalon. Efferents(More)