Maeda Y, Ueda H, Hara T, Kazami J, Kawano G, Suzuki E, Nagamune T
University of Tokyo, Japan.
Biotechniques. 1996 Jan;20(1):116-21. doi: 10.2144/96201rr01.
We have designed and constructed a novel chimeric protein that consisted of a single domain of protein A and luciferase derived from sea-firefly Vargula hilgendorfii with the goal of obtaining a heterofunctional immunological tool. The structural gene of luciferase was fused to the 3' terminus of the D domain gene of protein A with/without a short linker of five amino acids. The resulting constructs under the transcriptional regulation of the Rous sarcoma virus (RSV) promoter, were expressed transiently in simian COS-1 and stably in Chinese hamster ovary (CHO) cells. The properties of the resultant chimeric protein were characterized. The results indicated that the dual properties of the chimeric protein could be retained only after the introduction of a linker of (Gly)4 Ser between the two conjugated moieties. Moreover, the chimeric protein was found to retain at least 50% of the specific activity as compared with the non-fused luciferase. The future prospect of the usage of this chimeric protein in the field of diagnostics was further evaluated by performing bioluminescent immunoassays.
我们设计并构建了一种新型嵌合蛋白，它由蛋白A的单个结构域和源自海萤（Vargula hilgendorfii）的荧光素酶组成，目的是获得一种具有多种功能的免疫学工具。荧光素酶的结构基因与蛋白A的D结构域基因的3'末端融合，融合时带有或不带有一个由五个氨基酸组成的短连接子。所得构建体在劳氏肉瘤病毒（RSV）启动子的转录调控下，在猴COS-1细胞中瞬时表达，在中国仓鼠卵巢（CHO）细胞中稳定表达。对所得嵌合蛋白的性质进行了表征。结果表明，只有在两个共轭部分之间引入（Gly）4 Ser连接子后，嵌合蛋白的双重性质才能得以保留。此外，发现该嵌合蛋白与未融合的荧光素酶相比，保留了至少50%的比活性。通过进行生物发光免疫测定，进一步评估了这种嵌合蛋白在诊断领域的应用前景。
