Department of Basic and Applied Biology, University of L'Aquila, Via Vetoio snc., Coppito, 67010 L'Aquila, Italy.
Protein Eng Des Sel. 2010 Feb;23(2):61-8. doi: 10.1093/protein/gzp070. Epub 2009 Nov 23.
A critical problem in studying ribosome-inactivating proteins (RIPs) lies in the very limited possibility to produce them in heterologous systems. In fact, their inherent toxicity for the producing organism nearly always prevents their recombinant expression. In this study, we designed, expressed and characterized an engineered form of the RIP saporin (SapVSAV), bearing a C-terminal extra sequence that is recognized and bound by the second PDZ domain from murine PTP-BL protein (PDZ2). The co-expression of SapVSAV and PDZ2 in Escherichia coli BL21 cells greatly enhances the production of the toxin in a soluble form. The increase of production was surprisingly not due to protection from bacterial intoxication, but may arise from a stabilization effect of PDZ2 on the toxin molecule during biosynthesis. We found that once purified, SapVSAV is stable but is not toxic to free ribosomes, while it is fully active against human cancer cells. This strategy of co-expression of a toxin moiety and a soluble PDZ domain may represent a new system to increase the production of recombinant toxic proteins and could allow the selection of new extra sequences to target PDZ domains inside specific mammalian cellular domains.
研究核糖体失活蛋白（RIPs）的一个关键问题在于，它们在异源系统中的生产可能性非常有限。事实上，它们对产生它们的生物体的固有毒性几乎总是阻止了它们的重组表达。在这项研究中，我们设计、表达和表征了一种工程形式的 RIP 相思豆毒素（SapVSAV），它带有一个 C 末端额外的序列，该序列被来自鼠 PTP-BL 蛋白（PDZ2）的第二个 PDZ 结构域识别和结合。SapVSAV 和 PDZ2 在大肠杆菌 BL21 细胞中的共表达大大提高了毒素在可溶性形式下的产生。产量的增加令人惊讶的不是由于防止细菌中毒，而是可能源于 PDZ2 在生物合成过程中对毒素分子的稳定作用。我们发现，一旦纯化，SapVSAV 是稳定的，但对游离核糖体没有毒性，而对人癌细胞则完全有效。这种共表达毒素部分和可溶性 PDZ 结构域的策略可能代表了一种增加重组毒性蛋白产量的新系统，并允许选择新的额外序列来靶向特定哺乳动物细胞区域内的 PDZ 结构域。
