Gleiter S, Stubenrauch K, Lilie H
Martin-Luther-Universität Halle, Institut für Biotechnologie, Germany.
Protein Sci. 1999 Dec;8(12):2562-9. doi: 10.1110/ps.8.12.2562.
Recent developments on virus-like particles have demonstrated their potential in transfecting eucaryotic cells. In the case of particles based on the major coat protein VP1 of polyoma virus, transfection occurs via binding of VP1 to sialic acids. Since sialic acid is present on almost every eucaryotic cell line, this results in an unspecific cell targeting. Generation of a cell-type specificity of this system would imply the presentation of a new function on the surface of VP1. To analyze whether a new functional protein can be placed on VP1, we inserted dihydrofolate reductase from Escherichia coli as a model protein. The effect of such an insertion on both VP1 and the inserted protein was investigated, respectively. The function of VP1, like the formation of pentameric capsomers and its ability to assemble into capsids, was not influenced by the insertion. The inserted dihydrofolate reductase showed major changes when compared to the wild-type form. The thermal stability of the enzyme was dramatically reduced in the fusion protein; nevertheless, the dihydrofolate reductase proved to be a fully active enzyme with only slightly increased K(M) values for its substrates. This model system provides the basis for further modifications of the VP1 protein to achieve an altered surface of VP1 with new properties.
病毒样颗粒的最新进展已证明其在转染真核细胞方面的潜力。以多瘤病毒主要衣壳蛋白VP1为基础的颗粒为例，转染是通过VP1与唾液酸结合实现的。由于几乎每个真核细胞系上都存在唾液酸，这导致了非特异性的细胞靶向。该系统细胞类型特异性的产生意味着在VP1表面呈现一种新功能。为了分析是否可以在VP1上放置一种新的功能蛋白，我们插入了来自大肠杆菌的二氢叶酸还原酶作为模型蛋白。分别研究了这种插入对VP1和插入蛋白的影响。VP1的功能，如五聚体壳粒的形成及其组装成衣壳的能力，不受插入的影响。与野生型形式相比，插入的二氢叶酸还原酶表现出重大变化。融合蛋白中该酶的热稳定性显著降低；然而，二氢叶酸还原酶被证明是一种完全有活性的酶，其底物的米氏常数（K(M)）仅略有增加。这个模型系统为进一步修饰VP1蛋白以实现具有新特性的VP1表面改变提供了基础。
