Hedhammar My, Hober Sophia
Department of Biotechnology, Royal Institute of Technology, Albanova University Center, Stockholm, Sweden.
J Chromatogr A. 2007 Aug 17;1161(1-2):22-8. doi: 10.1016/j.chroma.2007.05.091. Epub 2007 Jun 3.
A positively charged protein domain, Z(basic), can be used as a general purification tag to achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography. To construct a protein domain usable for ion-exchange chromatography, the surface of protein Z was engineered to be highly charged, which allowed for selective capture of target proteins on a cation-exchanger at physiological pH values. Interestingly, the novel domain, denoted Z(basic), was shown to be selective also under denaturing conditions and could preferably be used for purification of proteins solubilised from inclusion bodies. Moreover, a flexible process for solid-phase refolding was developed, using Z(basic) as a reversible linker to the cation-exchanger resin. This procedure has the inherited advantage of combining purification and refolding into a single step and still enabling elution of a concentrated product in a suitable buffer. This article summarizes development and use of the Z(basic) tag in small and pilot-plant-scale downstream processing.
带正电荷的蛋白质结构域Z(basic)，可用作通用纯化标签，通过阳离子交换色谱法高效回收重组表达的目标蛋白。为构建适用于离子交换色谱的蛋白质结构域，对蛋白质Z的表面进行工程改造，使其带电量很高，从而能够在生理pH值条件下在阳离子交换剂上选择性捕获目标蛋白。有趣的是，新的结构域Z(basic)在变性条件下也具有选择性，并且特别适用于纯化从包涵体中溶解出来的蛋白质。此外，还开发了一种灵活的固相重折叠方法，使用Z(basic)作为与阳离子交换树脂的可逆连接体。该方法具有将纯化和重折叠合并为一步的固有优势，并且仍能在合适的缓冲液中洗脱浓缩产物。本文总结了Z(basic)标签在小规模和中试规模下游加工中的开发和应用。
