Two-step method to isolate target recombinant protein from co-purified bacterial contaminant SlyD after immobilised metal affinity chromatography

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. (c) 2007 Elsevier B.V. All rights reserved.