BACKGROUND: Hyaluronidase (Hya) is one of several allergens in honeybee venom. Its cDNA sequence was recently described. OBJECTIVE: We sought to express recombinant Hya in prokaryotic and eukaryotic systems and to compare it with natural (n)Hya for biologic activity. METHODS: In Escherichia coli Hya was produced as inclusion body 6 x His-fusion protein. In baculovirus-infected insect cells expression was obtained by cotransfection of linearized Bac-N-Blue DNA and pMelBac transfer vector into Spodoptera frugiperda cells. RESULTS: Enzymatic activity of Hya from the baculovirus system was equal to nHya, and that of the enzyme expressed in E. coli was only 20% to 30% of nHya. In vitro IgE binding was similar in nHya and the enzyme from baculovirus but markedly lower in Hya expressed in E. coli. CONCLUSIONS: Biologic activity of Hya expressed in baculovirus-infected insect cells was comparable with that of the natural enzyme, indicating a native-like conformation of the recombinant protein. In contrast, the enzyme expressed in E. coli as an inclusion-body protein and reconstituted in vitro reached only 20% to 30% of the activity of nHya.
BACKGROUND:Hyaluronidase (Hya) is one of several allergens in honeybee venom. Its cDNA sequence was recently described. OBJECTIVE: We sought to express recombinant Hya in prokaryotic and eukaryotic systems and to compare it with natural (n)Hya for biologic activity. METHODS: In Escherichia coliHya was produced as inclusion body 6 x His-fusion protein. In baculovirus-infected insect cells expression was obtained by cotransfection of linearized Bac-N-Blue DNA and pMelBac transfer vector into Spodoptera frugiperda cells. RESULTS: Enzymatic activity of Hya from the baculovirus system was equal to nHya, and that of the enzyme expressed in E. coli was only 20% to 30% of nHya. In vitro IgE binding was similar in nHya and the enzyme from baculovirus but markedly lower in Hya expressed in E. coli. CONCLUSIONS: Biologic activity of Hya expressed in baculovirus-infected insect cells was comparable with that of the natural enzyme, indicating a native-like conformation of the recombinant protein. In contrast, the enzyme expressed in E. coli as an inclusion-body protein and reconstituted in vitro reached only 20% to 30% of the activity of nHya.
Authors: Amilcar Perez-Riverol; Alexis Musacchio-Lasa; Luis Gustavo Romani Fernandes; Jose Roberto Aparecido Dos Santos-Pinto; Franciele Grego Esteves; Murilo Luiz Bazon; Ricardo de Lima Zollner; Mario Sergio Palma; Márcia Regina Brochetto-Braga Journal: 3 Biotech Date: 2020-04-27 Impact factor: 2.406
Authors: David I Bernstein; Hana M El Sahly; Wendy A Keitel; Mark Wolff; Gina Simone; Claire Segawa; Susan Wong; Daniel Shelly; Neal S Young; Walla Dempsey Journal: Vaccine Date: 2011-07-30 Impact factor: 3.641
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Authors: Simon Blank; Henning Seismann; Mareike McIntyre; Markus Ollert; Sara Wolf; Frank I Bantleon; Edzard Spillner Journal: PLoS One Date: 2013-04-23 Impact factor: 3.240