title: Superior biologic activity of the recombinant bee venom allergen hyaluronidase expressed in baculovirus-infected insect cells as compared with Escherichia coli.
abstract: 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.
authors.affiliation: Zieglerspital and Institute of Biochemistry, University of Bern, Switzerland.
authors.forename: L N
authors.initials: LN
authors.lastname: Soldatova
authors.forename: R
authors.initials: R
authors.lastname: Crameri
authors.forename: M
authors.initials: M
authors.lastname: Gmachl
authors.forename: D M
authors.initials: DM
authors.lastname: Kemeny
authors.forename: M
authors.initials: M
authors.lastname: Schmidt
authors.forename: M
authors.initials: M
authors.lastname: Weber
authors.forename: U R
authors.initials: UR
authors.lastname: Mueller
mesh_terms: D000485:Allergens / Q000235:genetics / Q000276:immunology*; D000818:Animals; D016367:Baculoviridae; D001514:Bee Venoms / Q000201:enzymology*; D002460:Cell Line; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D006639:Histidine; D006821:Hyaluronoglucosaminidase / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D018411:Spodoptera
chemical_list: D000485:Allergens; D001514:Bee Venoms; D011993:Recombinant Fusion Proteins; D006639:Histidine; D006821:Hyaluronoglucosaminidase