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J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Jun 15;853(1-2):314-9. Epub 2007 Apr 8.

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

Author information

1
OncImmune Limited, Clinical Sciences Building, Nottingham City Hospital, Hucknall Road, and Tumor Immunology Group, University of Nottingham NG5 1PB, UK. celine.parsy@nottingham.ac.uk

Abstract

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.

PMID:
17459787
DOI:
10.1016/j.jchromb.2007.03.046
[Indexed for MEDLINE]

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