Parsy Céline B, Chapman Caroline J, Barnes Antony C, Robertson John F, Murray Andrea
OncImmune Limited, Clinical Sciences Building, Nottingham City Hospital, Hucknall Road, and Tumor Immunology Group, University of Nottingham NG5 1PB, UK.
J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Jun 15;853(1-2):314-9. doi: 10.1016/j.jchromb.2007.03.046. Epub 2007 Apr 8.
As part of a study to purify the internal domain of HER2 (ICD) from recombinant expression, through metal immobilised affinity chromatography (IMAC), we encountered a contaminant, SlyD, a 29 kDa native E. coli protein. SlyD is a recurrent contaminant, with a histidine rich domain enabling binding to IMAC columns and thus co-elution with the target protein. Research has been carried out on this protein and its purification, however, no work mentions how to treat it as a true contaminant or describe procedures to isolate it from target proteins. In this report, we described a two-step chromatographic method for the purification of ICD, including IMAC as a capture step and size exclusion chromatography (SEC) as a polishing step. IMAC allowed us to purify ICD from bacterial crude with SlyD co-eluting. SEC then allowed us to resolve ICD from SlyD and achieve a purity greater than 95% for ICD. However, this method has been developed to accommodate any protein whose molecular weight is different enough from SlyD to be separated by SEC.
作为从重组表达中纯化HER2胞内结构域（ICD）研究的一部分，我们采用金属固定化亲和色谱法（IMAC）进行纯化，在此过程中遇到了一种污染物，即SlyD，它是一种29 kDa的天然大肠杆菌蛋白。SlyD是一种常见的污染物，其富含组氨酸的结构域能够与IMAC柱结合，从而与目标蛋白共洗脱。关于这种蛋白及其纯化已有相关研究，但没有文献提及如何将其作为真正的污染物进行处理，也没有描述从目标蛋白中分离它的方法。在本报告中，我们描述了一种用于纯化ICD的两步色谱法，包括以IMAC作为捕获步骤，以尺寸排阻色谱法（SEC）作为精制步骤。IMAC使我们能够从含有共洗脱的SlyD的细菌粗提物中纯化ICD。然后，SEC使我们能够将ICD与SlyD分离，并使ICD的纯度达到95%以上。然而，该方法已被开发用于处理任何分子量与SlyD有足够差异从而能够通过SEC分离的蛋白。
