Department of Biotechnology, University of Natural Resources and Applied Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria.
J Chromatogr A. 2009 Nov 27;1216(48):8460-9. doi: 10.1016/j.chroma.2009.10.012. Epub 2009 Oct 12.
Refolding of proteins must be performed under very dilute conditions to overcome the competing aggregation reaction, which has a high reaction order. Refolding on a chromatography column partially prevents formation of the intermediate form prone to aggregation. A chromatographic refolding procedure was developed using an autoprotease fusion protein with the mutant EDDIE from the N(pro) autoprotease of pestivirus. Upon refolding, self-cleavage generates a target peptide with an authentic N-terminus. The refolding process was developed using the basic 1.8-kDa peptide sSNEVi-C fused to the autoprotease EDDIE or the acidic peptide pep6His, applying cation and anion exchange chromatography, respectively. Dissolved inclusion bodies were loaded on cation exchange chromatographic resins (Capto S, POROS HS, Fractogel EMD SO(3)(-), UNOsphere S, SP Sepharose FF, CM Sepharose FF, S Ceramic HyperD F, Toyopearl SP-650, and Toyopearl MegaCap II SP-550EC). A conditioning step was introduced in order to reduce the urea concentration prior to the refolding step. Refolding was initiated by applying an elution buffer containing a high concentration of Tris-HCl plus common refolding additives. The actual refolding process occurred concurrently with the elution step and was completed in the collected fraction. With Capto S, POROS HS, and Fractogel SO(3)(-), refolding could be performed at column loadings of 50mg fusion protein/ml gel, resulting in a final eluate concentration of around 10-15 mg/ml, with refolding and cleavage step yields of around 75%. The overall yield of recovered peptide reached 50%. Similar yields were obtained using the anion exchange system and the pep6His fusion peptide. This chromatographic refolding process allows processing of fusion peptides at a concentration range 10- to 100-fold higher than that observed for common refolding systems.
蛋白质的复性必须在非常稀释的条件下进行，以克服具有高反应级数的聚集反应。在色谱柱上复性部分防止了易于聚集的中间形式的形成。使用来自瘟病毒 N（pro）自蛋白酶的突变 EDDIE 的自蛋白酶融合蛋白开发了一种色谱复性程序。复性时，自我切割产生具有真实 N 末端的靶肽。该复性过程是使用碱性 1.8 kDa 肽 sSNEVi-C 与自蛋白酶 EDDIE 或酸性肽 pep6His 融合开发的，分别应用阳离子和阴离子交换色谱法。溶解的包涵体加载到阳离子交换色谱树脂（Capto S、POROS HS、Fractogel EMD SO（3）（-）、UNOsphere S、SP Sepharose FF、CM Sepharose FF、S Ceramic HyperD F、Toyopearl SP-650 和 Toyopearl MegaCap II SP-550EC）上。引入了一个调节步骤，以便在复性步骤之前降低脲浓度。复性通过施加含有高浓度 Tris-HCl 加常见复性添加剂的洗脱缓冲液来启动。实际的复性过程与洗脱步骤同时发生，并在收集的馏分中完成。使用 Capto S、POROS HS 和 Fractogel SO（3）（-），可以在 50mg 融合蛋白/ml 凝胶的柱载量下进行复性，导致最终洗脱液浓度约为 10-15mg/ml，复性和切割步骤产率约为 75%。回收肽的总产率达到 50%。使用阴离子交换系统和 pep6His 融合肽也获得了类似的产率。这种色谱复性过程允许在比常见复性系统观察到的浓度高 10 到 100 倍的范围内处理融合肽。
