Agarwal Rakhi, Eswaramoorthy Subramaniam, Kumaran Desigan, Dunn John J, Swaminathan Subramanyam
Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.
Protein Expr Purif. 2004 Mar;34(1):95-102. doi: 10.1016/j.pep.2003.10.017.
The catalytic activity of the highly potent botulinum neurotoxins are confined to their N-terminal light chains ( approximately 50kDa). A full-length light chain for the type E neurotoxin with a C-terminal 6x His-tag, BoNT/E-LC, has been cloned in a pET-9c vector and over-expressed in BL21 (DE3) cells. BoNT/E-LC was purified to homogeneity by affinity chromatography on Ni-NTA agarose followed by exclusion chromatography using a Superdex-75 sizing column. The purified protein has very good solubility and can be stored stably at -20 degrees C; however, it seems to undergo auto-proteolysis when stored at temperature #10878;4-10 degrees C. BoNT/E-LC is active on its natural substrate, the synaptosomal associated 25kDa protein, SNAP-25, indicating that it retains a native-like conformation and therefore can be considered as a useful tool in studying the structure/function of the catalytic light chain. Recombinant BoNT/E-LC has been crystallized under five different conditions and at various pHs. Crystals diffract to better than 2.1A.
高效力肉毒杆菌神经毒素的催化活性局限于其N端轻链（约50kDa）。带有C端6x组氨酸标签的E型神经毒素全长轻链BoNT/E-LC已克隆到pET-9c载体中，并在BL21(DE3)细胞中过量表达。通过在Ni-NTA琼脂糖上进行亲和层析，随后使用Superdex-75尺寸排阻柱进行排阻层析，将BoNT/E-LC纯化至同质。纯化后的蛋白具有很好的溶解性，可在-20℃稳定保存；然而，在4-10℃保存时似乎会发生自催化降解。BoNT/E-LC对其天然底物突触体相关25kDa蛋白SNAP-25具有活性，表明它保留了类似天然的构象，因此可被视为研究催化轻链结构/功能的有用工具。重组BoNT/E-LC已在五种不同条件和不同pH值下结晶。晶体衍射分辨率优于2.1埃。
