Jung H C, Park J H, Park S H, Lebeault J M, Pan J G
Korea Research Institute of Bioscience and Biotechnology (KRIBB), KIST, Yusong, Taejon, Korea.
Enzyme Microb Technol. 1998 Apr;22(5):348-54. doi: 10.1016/s0141-0229(97)00224-x.
Ice-nucleation protein (INP), an outer membrane protein from Pseudomonas syringae, is able to catalyze the ice crystal formation of supercooled water. It was exploited for anchoring of Bacillus subtilis carboxymethylcellulase (CMCase) on the surface of Escherichia coli. A surface anchoring vector, pGINP21M, was created that contains the multicloning sites including BamHI, SmaI and EcoRI at the end of the 3' flanking region encoding the C-terminus of INP instead of the stop codon for subcloning the foreign genes. The CMCase gene was in-frame subcloned for making INP-CMCase fusion proteins. The ability of this vector for directing the actual synthesis of INP-CMCase fusion proteins was confirmed by Western blotting analysis. CMCase targeted on the surface of cells was verified by measuring whole cell CMCase activity and ice-nucleation activity. CMCase activity was mainly detected on the cell surface whereas no enzyme activity was detected in the culture supernatant. Ice-nucleation activity was also maintained even if an INP-CMCase hybrid was made. This means that the fusion protein is functionally expressed and has its biological conformation on the surface. INP-CMCase fusion proteins were stable in the stationary phase. INP deleted of the repeating domain, thus producing no ice-nucleation activity, could also direct CMCase on the cell surface. This suggests that it has the secretion and targeting signal to the outer membrane.
冰核蛋白（INP）是丁香假单胞菌的一种外膜蛋白，能够催化过冷水的冰晶形成。它被用于将枯草芽孢杆菌羧甲基纤维素酶（CMCase）锚定在大肠杆菌表面。构建了一个表面锚定载体pGINP21M，该载体在编码INP C端的3'侧翼区域末端包含多克隆位点，包括BamHI、SmaI和EcoRI，取代了用于亚克隆外源基因的终止密码子。将CMCase基因进行读码框亚克隆以制备INP-CMCase融合蛋白。通过蛋白质免疫印迹分析证实了该载体指导实际合成INP-CMCase融合蛋白的能力。通过测量全细胞CMCase活性和冰核活性验证了靶向细胞表面的CMCase。CMCase活性主要在细胞表面检测到，而在培养上清液中未检测到酶活性。即使制备了INP-CMCase杂种，冰核活性也得以维持。这意味着融合蛋白在功能上得到表达并在表面具有其生物学构象。INP-CMCase融合蛋白在稳定期是稳定的。缺失重复结构域从而不产生冰核活性的INP也能将CMCase引导至细胞表面。这表明它具有向外膜的分泌和靶向信号。
