Superior biologic activity of the recombinant bee venom allergen hyaluronidase expressed in baculovirus-infected insect cells as compared with Escherichia coli

• Published in: Journal of allergy and clinical immunology Vol. 101; no. 5; pp. 691 - 698
• Main Authors: Soldatova, Lyudmila N., Crameri, Reto, Gmachl, Michael, Kemeny, David M., Schmidt, Margit, Weber, Margrit, Mueller, Ulrich R.
• Format: Journal Article
• Language: English
• Published: New York, NY Mosby, Inc 01.05.1998; Elsevier
• Subjects: Allergens - genetics; Allergens - immunology; Animals; Apis mellifera; Baculoviridae; Baculovirus; Bee venom allergy; Bee Venoms - enzymology; Biological and medical sciences; Cell Line; enzymatic activity; Escherichia coli; Escherichia coli - metabolism; Gene Expression; Genetic Vectors; Histidine; hyaluronidase; Hyaluronoglucosaminidase - genetics; Hyaluronoglucosaminidase - immunology; Hyaluronoglucosaminidase - isolation & purification; Immunopathology; Immunotherapy (general aspects); Medical sciences; Protein Folding; recombinant allergens; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - isolation & purification; specific IgE; Spodoptera; hyaluronidase; BvHya; PLA; TBS; recombinant allergens; EcHya; specific IgE; Hya; SDS-PAGE; baculovirus; Bee venom allergy; enzymatic activity; PCR; Human; Hyaluronidase; Allergy; Immunopathology; Desensitization; Escherichia coli; Insecta; Method; Biological activity; Treatment; Arthropoda; Immunotherapy; Venom; Bacteria; Manufacturing; Recombinant protein; Invertebrata; Comparative study; Allergen; Enterobacteriaceae
• ISSN: 0091-6749; 1097-6825
• DOI: 10.1016/S0091-6749(98)70179-4

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×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.(J Allergy Clin Immunol 1998;101:691-8.)