Structural characteristics of hen egg ovalbumin expressed in yeast Pichia pastoris

The recombinant ovalbumin (OVA) produced in yeast Pichia pastoris was purified from the culture medium by anion exchange chromatography, and its structural characteristics were compared with those of hen egg OVA, mainly from the point of view of post-translational modification. The expressed OVA con...

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Veröffentlicht in:Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2005-04, Vol.69 (4), p.755-761
Hauptverfasser: Ito, K. (Yamaguchi Univ. (Japan). Faculty of Agriculture), Matsudomi, N
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Sprache:eng
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Zusammenfassung:The recombinant ovalbumin (OVA) produced in yeast Pichia pastoris was purified from the culture medium by anion exchange chromatography, and its structural characteristics were compared with those of hen egg OVA, mainly from the point of view of post-translational modification. The expressed OVA consisted of two molecular species immmuno-reactive with antibody for hen egg OVA. The two molecular species, 45 and 47 kDa in molecular size, were thought to correspond to mono-glycosylated form and di-glycosy-lated form respectively. The non-glycosylated form was not produced in the system. The other post-translational modifications (N-terminal acetylation and phosphorylation) observed in hen egg OVA were not detected in either of the molecular species. The two recombinant proteins displayed almost exactly the same circular dichroism and intrinsic tryptophan fluorescence spectra as hen egg OVA. The melting temperature, Tsub(m), which was determined from the thermal unfolding curve, was almost identical in the two recombinant proteins, despite the difference in glycosylation levels, while it decreased by about 2.5 deg C as compared with that of hen egg OVA (77.3 deg C). These data indicate that the additional glycosylation to Asn-311 in the recombinant protein does not affect protein conformation or thermo-stability.
ISSN:0916-8451
1347-6947
DOI:10.1271/bbb.69.755