Tang Jin-Bao, Zhu Peng, Yang Hong-ming, Sun Li-Min, Song Shu-liang, Ji Ai-guo
Department of Basic Medicine, Weifang Medical University, WeiFang 261042, People's Republic of China.
Biotechnol Lett. 2008 Aug;30(8):1409-14. doi: 10.1007/s10529-008-9714-5. Epub 2008 Apr 16.
We constructed a fusion protein ZZ-EGFP by fusing the ZZ domains of staphylococcal protein A (SpA) and enhanced green fluorescent protein (EGFP). ZZ-EGFP was secreted in the yeast, Pichia pastoris, with a hexahistidine tag. Its expression level was determined by measuring the fluorescence of EGFP. When the recombinant yeast cells in shake-flasks were induced with 0.5% methanol for 96 h, a maximum yield of 115 mg ZZ-EGFP/l was obtained. The resulting ZZ-EGFP fusion protein retained immunoglobulin G (IgG)-binding capacity and EGFP fluorescence. ZZ-EGFP was then used in immunofluorescence assays for detecting antinuclear antibodies (ANA); it produced a good signal that was comparable in its brightness and fluorescence pattern to that generated with fluorescein isothiocyanate (FITC)-labelled anti-human IgG. Thus, ZZ-EGFP showed great potential in immunological applications due to its ability to bind to various IgG from different animal sources.
我们通过融合葡萄球菌蛋白A（SpA）的ZZ结构域和增强型绿色荧光蛋白（EGFP）构建了一种融合蛋白ZZ-EGFP。ZZ-EGFP在毕赤酵母中分泌，并带有一个六组氨酸标签。其表达水平通过测量EGFP的荧光来确定。当摇瓶中的重组酵母细胞用0.5%甲醇诱导96小时时，ZZ-EGFP的最高产量达到115 mg/l。所得的ZZ-EGFP融合蛋白保留了免疫球蛋白G（IgG）结合能力和EGFP荧光。然后将ZZ-EGFP用于免疫荧光分析以检测抗核抗体（ANA）；它产生了良好的信号，其亮度和荧光模式与异硫氰酸荧光素（FITC）标记的抗人IgG产生的信号相当。因此，由于ZZ-EGFP能够结合来自不同动物来源的各种IgG，它在免疫学应用中显示出巨大潜力。
