Kadkhodayan S, Knapp M S, Schmidt J J, Fabes S E, Rupp B, Balhorn R
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94551, USA.
Protein Expr Purif. 2000 Jun;19(1):125-30. doi: 10.1006/prep.2000.1227.
A truncated but functional form of the botulinum neurotoxin A light chain (Tyr 9-Leu 415) has been cloned into the three bacterial expression vectors, pET 28, pET 30, and PGEX-2T, and produced as fusion proteins. This 406-amino-acid light chain was expressed with 1 six-histidine tag (LC-pET28), 2 six histidine tags and a S-tag (LC-pET30), or a six-histidine tag and a glutathione S-transferase tag (LC-pGEX-2T). The three fusion proteins have been overexpressed in Escherichia coli, purified in a soluble form, and tested for protease activity. All three recombinant proteins were found to have similar enzymatic activity, comparable to the light chain purified from the whole toxin. The LC-pET30 protein was the most soluble and stable of the three fusion proteins, and it could be purified using a one-step affinity chromatography protocol. The purified protein was determined to be 98% pure as assessed by SDS-polyacrylamide gel. This protein has been crystallized and initial X-ray data show that the crystals diffract to 1.8 A.
肉毒杆菌神经毒素A轻链（Tyr 9-Leu 415）的截短但具有功能的形式已被克隆到三种细菌表达载体pET 28、pET 30和PGEX-2T中，并作为融合蛋白进行表达。这个由406个氨基酸组成的轻链与1个六组氨酸标签（LC-pET28）、2个六组氨酸标签和1个S标签（LC-pET30）或1个六组氨酸标签和1个谷胱甘肽S-转移酶标签（LC-pGEX-2T）一起表达。这三种融合蛋白已在大肠杆菌中过表达，以可溶形式纯化，并检测了蛋白酶活性。发现所有三种重组蛋白都具有相似的酶活性，与从全毒素中纯化的轻链相当。LC-pET30蛋白是三种融合蛋白中最易溶且最稳定的，它可以通过一步亲和层析方案进行纯化。经SDS-聚丙烯酰胺凝胶评估，纯化后的蛋白纯度为98%。该蛋白已结晶，初步的X射线数据表明晶体的衍射分辨率达到1.8埃。
