Noronha S, Kaufman J, Shiloach J
Biotechnology Unit, NIDDK, NIH, Bethesda, MD 20892, USA.
Bioseparation. 1999;8(1-5):145-51.
Expression of recombinant proteins with poly-histidine tags enables their convenient capture and purification using immobilized metal affinity chromatography (IMAC). The 6 x His-tagged protein binds to a chelating resin charged with metal ions such as Ni2+, Cu2+ or Zn2+, and can therefore be separated from proteins which have lower, or no, affinity for the resin. Two recombinant proteins, a malaria transmission-blocking vaccine candidate secreted extracellularly by S. cerevisiae and a modified diphtheria toxin produced intracellularly by E. coli, were expressed with 6 x His tags and could therefore be purified using IMAC. In an effort to further simplify the initial capture of these proteins, an expanded bed adsorption technique using a chelating resin (Streamline Chelating) was introduced. It was possible to capture the intracellular diphtheria protein from E. coli directly after cell lysis, without prior centrifugation or filtration. The extracellular malaria vaccine candidate was also directly captured from a high cell density yeast culture. Detailed information on the experimental work performed, and the capture processes developed, is provided.
带有多组氨酸标签的重组蛋白的表达，使得使用固定化金属亲和色谱法（IMAC）对其进行便捷的捕获和纯化成为可能。带有6×组氨酸标签的蛋白会与负载有金属离子（如Ni2+、Cu2+或Zn2+）的螯合树脂结合，因此可以与那些对该树脂亲和力较低或没有亲和力的蛋白分离开来。两种重组蛋白，一种由酿酒酵母细胞外分泌的疟疾传播阻断疫苗候选物，以及一种由大肠杆菌在细胞内产生的修饰白喉毒素，都带有6×组氨酸标签进行表达，因此可以使用IMAC进行纯化。为了进一步简化这些蛋白的初始捕获过程，引入了一种使用螯合树脂（Streamline Chelating）的扩张床吸附技术。在细胞裂解后，无需事先离心或过滤，就可以直接从大肠杆菌中捕获细胞内的白喉蛋白。细胞外的疟疾疫苗候选物也可以直接从高细胞密度的酵母培养物中捕获。文中提供了关于所进行的实验工作以及所开发的捕获过程的详细信息。
