title: Secreted production of a custom-designed, highly hydrophilic gelatin in Pichia pastoris.
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.
authors.affiliation: Agrotechnological Research Institute (ATO BV), Bornsesteeg 59, 6708 PD Wageningen, The Netherlands. m.w.t.werten@ato.wag-ur.nl
authors.forename: M W
authors.initials: MW
authors.lastname: Werten
authors.forename: W H
authors.initials: WH
authors.lastname: Wisselink
authors.forename: T J
authors.initials: TJ
authors.lastname: Jansen-van den Bosch
authors.forename: E C
authors.initials: EC
authors.lastname: de Bruin
authors.forename: F A
authors.initials: FA
authors.lastname: de Wolf
mesh_terms: D001483:Base Sequence; D002942:Circular Dichroism; D015195:Drug Design; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D004951:Esterification; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005780:Gelatin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D055672:Static Electricity; D013500:Surface Tension; D014169:Transformation, Bacterial
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D005780:Gelatin