Kamiya Noriho, Doi Satoshi, Tominaga Jo, Ichinose Hirofumi, Goto Masahiro
Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 6-10-1 Hakozaki, Fukuoka, Japan.
Biomacromolecules. 2005 Jan-Feb;6(1):35-8. doi: 10.1021/bm0494895.
An enzymatic method for covalent and site-specific immobilization of recombinant proteins on a plastic surface was explored. Using Escherichia coli alkaline phosphatase (AP) with a specific peptide tag (MKHKGS) genetically incorporated at the N-terminus as a model (NK-AP), microbial transglutaminase (MTG)-mediated protein immobilization was demonstrated. To generate a reactive surface for MTG, a 96-well polystyrene microtiter plate was physically coated with casein, a good MTG substrate. Successful immobilization of recombinant AP to the nanolayer of casein on the surface of the microtiter plate was verified by the detection of enzymatic activity. Since little activity was observed when wild-type AP was used, immobilization of NK-AP was likely directed by the specific peptide tag. When polymeric casein prepared by MTG was used as a matrix on the plate, the loading capacity of AP was increased about 2-fold compared to when casein was used as the matrix. Transglutaminase-mediated site-specific posttranslational modification of proteins offers one way of generating a variety of protein-based solid formulations for biotechnological applications.
探索了一种将重组蛋白共价且位点特异性固定在塑料表面的酶法。以在N端基因融合有特定肽标签（MKHKGS）的大肠杆菌碱性磷酸酶（AP）作为模型（NK-AP），证明了微生物转谷氨酰胺酶（MTG）介导的蛋白固定化。为了生成MTG的反应性表面，用MTG的良好底物酪蛋白对96孔聚苯乙烯微量滴定板进行物理包被。通过检测酶活性验证了重组AP成功固定在微量滴定板表面的酪蛋白纳米层上。由于使用野生型AP时观察到的活性很低，NK-AP的固定化可能是由特定肽标签引导的。当将MTG制备的聚合酪蛋白用作板上的基质时，与使用酪蛋白作为基质相比，AP的负载能力提高了约2倍。转谷氨酰胺酶介导的蛋白质位点特异性翻译后修饰为生物技术应用生成各种基于蛋白质的固体制剂提供了一种方法。
