Laboratory for Biosynthesis and Biotransformation, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
N Biotechnol. 2013 Jan 25;30(2):250-4. doi: 10.1016/j.nbt.2011.09.005. Epub 2011 Oct 1.
Recombinant protein production significantly improved in the past three decades. Novel expression systems were developed, growth conditions optimised and the technology and thus monitoring and analysis significantly enhanced. However, the studies of bacterial cell disruption were more or less neglected. The existing methods were acceptable until the final product of protein production was soluble and pure protein. However recently, inclusion bodies (IBs) as whole protein particles were also recognised as the final product. Classical methods for bacterial cell disruption are therefore not always suitable, sufficient or even appropriate for isolation of such particulate material. Some of the currently existing methods for bacterial cell disruption were recognised as damaging for the structure of IBs, while sonication was even found harmful for the recombinant protein. The powers needed for disruption of the bacterial cells damage the recombinant proteins and thus their biological activity significantly reduces. Furthermore, the classical isolation methods enable disruption of majority of the bacterial cells and this is enough for isolation of soluble proteins, yet it is not adequate for isolation of particulate material. While remaining bacterial cells sediment together with the IBs, they represent impurity. The need for isolation of cell-free IBs was therefore revealed in the recent studies, because only pure IBs can be used as nanoparticles in further biomedical applications. Therefore it is time to consider, redesign, optimise or even develop new alternative methods that would enable isolation of pure, structurally intact and biologically active particles. Two such alternative methods that enable isolation of bacterial free, active protein particles were developed recently.
在过去的三十年中，重组蛋白的生产有了显著的改进。新的表达系统被开发出来，生长条件得到了优化，技术因此得到了显著的提升，监测和分析也得到了加强。然而，细菌细胞破碎的研究或多或少被忽视了。直到蛋白质生产的最终产物是可溶性的纯蛋白，现有的方法才是可以接受的。然而，最近，包含体（IBs）作为完整的蛋白质颗粒也被认为是最终产物。因此，经典的细菌细胞破碎方法并不总是适合、充分或甚至适用于分离这种颗粒物质。一些现有的细菌细胞破碎方法被认为对 IBs 的结构有破坏作用，而超声处理甚至被发现对重组蛋白有害。用于破坏细菌细胞的能量会显著损害重组蛋白的生物活性。此外，经典的分离方法能够破坏大多数细菌细胞，这对于分离可溶性蛋白质已经足够，但对于分离颗粒物质则不够。由于剩余的细菌细胞与 IBs 一起沉淀，它们代表了杂质。因此，最近的研究揭示了分离无细胞 IBs 的需求，因为只有纯 IBs 才能在进一步的生物医学应用中用作纳米颗粒。因此，现在是时候考虑、重新设计、优化甚至开发新的替代方法，以实现纯、结构完整和具有生物活性的颗粒的分离。最近开发了两种能够分离无细菌、活性蛋白颗粒的替代方法。
