Secreted production of a custom-designed, highly hydrophilic gelatin in Pichia pastoris

A custom-designed, highly hydrophilic gelatin was produced in Pichia pastor is. Secreted production levels in single-copy transformants were in the ran ge 3-6 g/l of clarified broth and purification to near homogeneity could be accomplished by differential ammonium sulfate precipitation. Despite the f act that gelatins are highly susceptible to proteolysis because of their un folded structure, the recombinant protein was shown to be fully intact by S DS-PAGE, N-terminal sequencing, gel filtration chromatography and mass spec trometry. Owing to its highly hydrophilic nature, the migration of the synt hetic gelatin in SDS-PAGE was severely delayed. Esterification of the carbo xylic amino acid side chains resulted in normal migration. The high polarit y of the synthetic gelatin also accounts for its negligible surface activit y in water at concentrations up to 5% (w/v), as determined by tensiometry. Circular dichroism spectrometry showed that the non-hydroxylated gelatin di d not form triple helices at 4 degreesC. The spectrum was even more represe ntative of the random coil conformation than the spectrum of natural nonhyd roxylated gelatins.