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.
Parsy
Céline B
CB
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
Chapman
Caroline J
CJ
Barnes
Antony C
AC
Robertson
John F
JF
Murray
Andrea
A
D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008670:Metals / Q000737:chemistry*; D008970:Molecular Weight; D019696:Peptidylprolyl Isomerase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D029968:Escherichia coli Proteins; D008670:Metals; D011994:Recombinant Proteins; C085606:SlyD protein, E coli; D019696:Peptidylprolyl Isomerase