Lindbladh C, Mosbach K, Bülow L
Department of Pure and Applied Biochemistry, University of Lund, Sweden.
J Immunol Methods. 1991 Mar 21;137(2):199-207. doi: 10.1016/0022-1759(91)90025-b.
The genes encoding staphylococcal protein A and bacterial luciferase (Vibrio harveyi) were fused in-frame in order to obtain a general marker enzyme for bioluminescent immunoassays. Two constructs were made where protein A was ligated to the first and the 12th amino acid residue, respectively, of the N terminus of the beta subunit of luciferase. Only the first fusion protein encoding the entire beta subunit was able to form an enzymatically active luciferase complex when expressed together with the alpha subunit. The fusion of protein A to luciferase did not notably alter the emitted wavelength spectrum or its stability to urea treatment. The fusion protein was found to retain at least 50% of the specific bioluminescent activity compared to native luciferase. In preliminary tests, this hybrid protein was shown to be useful in bioluminescent immunoassays.
为了获得用于生物发光免疫分析的通用标记酶，将编码葡萄球菌蛋白A和细菌荧光素酶（哈维弧菌）的基因进行读码框融合。构建了两种融合体，其中蛋白A分别连接到荧光素酶β亚基N端的第一个和第十二个氨基酸残基上。只有编码完整β亚基的第一个融合蛋白在与α亚基共同表达时能够形成具有酶活性的荧光素酶复合物。蛋白A与荧光素酶的融合并没有显著改变发射的波长光谱或其对尿素处理的稳定性。与天然荧光素酶相比，发现融合蛋白保留了至少50%的特异性生物发光活性。在初步试验中，这种杂交蛋白被证明可用于生物发光免疫分析。
