Secreted production of a custom-designed, highly hydrophilic gelatin in Pichia pastoris |
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Authors: | Werten Marc W T; Wisselink Wouter H; Jansen-van den Bosch Tanja J; de Bruin Eric C; de Wolf Frits A |
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Affiliation: | Agrotechnological Research Institute (ATO BV), Bornsesteeg 59, 6708 PD Wageningen, The Netherlands |
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Abstract: | A custom-designed, highly hydrophilic gelatin was produced in
Pichia pastoris
. Secreted production levels in single-copytransformants were in the range 36 g/l of clarified brothand purification to near homogeneity could be accomplished bydifferential ammonium sulfate precipitation. Despite the factthat gelatins are highly susceptible to proteolysis becauseof their unfolded structure, the recombinant protein was shownto be fully intact by SDSPAGE, N-terminal sequencing,gel filtration chromatography and mass spectrometry. Owing toits highly hydrophilic nature, the migration of the syntheticgelatin in SDSPAGE was severely delayed. Esterificationof the carboxylic amino acid side chains resulted in normalmigration. The high polarity of the synthetic gelatin also accountsfor its negligible surface activity in water at concentrationsup to 5% (w/v), as determined by tensiometry. Circular dichroismspectrometry showed that the non-hydroxylated gelatin did notform triple helices at 4°C. The spectrum was even more representativeof the random coil conformation than the spectrum of naturalnon-hydroxylated gelatins. |
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