Soldatova L N, Crameri R, Gmachl M, Kemeny D M, Schmidt M, Weber M, Mueller U R
Zieglerspital and Institute of Biochemistry, University of Bern, Switzerland.
J Allergy Clin Immunol. 1998 May;101(5):691-8. doi: 10.1016/S0091-6749(98)70179-4.
Hyaluronidase (Hya) is one of several allergens in honeybee venom. Its cDNA sequence was recently described.
We sought to express recombinant Hya in prokaryotic and eukaryotic systems and to compare it with natural (n)Hya for biologic activity.
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.
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.
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.
透明质酸酶（Hya）是蜜蜂毒液中的几种过敏原之一。其cDNA序列最近已被描述。
我们试图在原核和真核系统中表达重组Hya，并将其与天然（n）Hya的生物活性进行比较。
在大肠杆菌中，Hya作为包涵体6×His融合蛋白产生。在杆状病毒感染的昆虫细胞中，通过将线性化的Bac-N-Blue DNA和pMelBac转移载体共转染到草地贪夜蛾细胞中来实现表达。
杆状病毒系统中Hya的酶活性与nHya相当，而在大肠杆菌中表达的酶活性仅为nHya的20%至30%。nHya和杆状病毒来源的酶在体外的IgE结合情况相似，但大肠杆菌中表达的Hya的IgE结合明显较低。
在杆状病毒感染的昆虫细胞中表达的Hya的生物活性与天然酶相当，表明重组蛋白具有类似天然的构象。相比之下，在大肠杆菌中作为包涵体蛋白表达并在体外重构的酶活性仅达到nHya活性的20%至30%。
