ABSTRACT
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 because of their unfolded structure, the recombinant protein was shown to be fully intact by SDS-PAGE, N-terminal sequencing, gel filtration chromatography and mass spectrometry. Owing to its highly hydrophilic nature, the migration of the synthetic gelatin in SDS-PAGE was severely delayed. Esterification of the carboxylic amino acid side chains resulted in normal migration. The high polarity of the synthetic gelatin also accounts for its negligible surface activity in water at concentrations up to 5% (w/v), as determined by tensiometry. Circular dichroism spectrometry showed that the non-hydroxylated gelatin did not form triple helices at 4 degrees C. The spectrum was even more representative of the random coil conformation than the spectrum of natural non-hydroxylated gelatins.
Subject(s)
Drug Design , Gelatin/chemistry , Pichia/genetics , Base Sequence , Circular Dichroism , Drug Stability , Electrophoresis, Polyacrylamide Gel , Esterification , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gelatin/genetics , Gelatin/metabolism , Molecular Sequence Data , Pichia/chemistry , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Solubility , Static Electricity , Surface Tension , Transformation, BacterialABSTRACT
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 the medium, even though gelatin is extremely susceptible to proteolysis due to its open, unfolded structure. Proteolytic cleavage at specific mono-arginylic sites, by a putative Kex2-like protease, could be successfully abolished by site-directed mutagenesis of these sites. Production levels as high as 14.8 g/l clarified both were obtained, using multicopy tranformants. To our knowledge, this represents the highest level of heterologous protein secretion reported to date for P. pastoris.
Subject(s)
Gelatin/metabolism , Pichia/metabolism , Proprotein Convertases , Recombinant Proteins/biosynthesis , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Collagen/metabolism , DNA/chemistry , DNA Primers/chemistry , Electrophoresis, Polyacrylamide Gel , Fermentation , Gelatin/analysis , Genetic Vectors/chemistry , Hydrogen-Ion Concentration , Molecular Sequence Data , Mutagenesis, Site-Directed , Pichia/genetics , Pichia/growth & development , Plasmids/chemistry , Recombinant Proteins/analysis , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Sequence Analysis , Subtilisins/chemistry , Transformation, GeneticABSTRACT
Homologous integration was studied in the common mushroom, Agaricus bisporus, using a plasmid (pHAG3-1) carrying the hygromycin-resistance gene and a 3.2-kb genomic fragment from A. bisporus. Homologous integration was found in 30-60% of the transformants obtained with pHAG3-1 linearized at three different positions within the homologous sequence, generating either blunt, 5'- or 3'-protruding ends. The genomic fragment was found to contain two homologous open reading frames in tandem, which showed 60% similarity to exo-beta-1,3-glucanases from Saccharomyces cerevisiae and Candida albicans. The level of the corresponding mRNA is low in the vegetative mycelium and relatively high in fruiting bodies. In the vegetative mycelium of a transformant with tandemly integrated pHAG3-1 plasmids at the homologous position, exoglucanase mRNA was strongly increased without any apparent effect on growth rate or morphology.
