Immobilized metal-ion affinity chromatography of recombinant Fab protein OPG C11 in the presence of EDTA–Mg(II)

• Published in: Journal of Chromatography A Vol. 978; no. 1; pp. 153 - 164
• Main Authors: Xiang, Hui, Wynn, Richard, Nguyen, Lien-Hanh T, Ross, O.Harold, Ahrens, Douglas P, O’Neil, Karyn T, Hollis, Gregory F, Patrick, Denis R
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 29.11.2002; Elsevier
• Subjects: Biological and medical sciences; Biotechnology; Chromatography, Affinity - methods; Edetic Acid - chemistry; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Immunoglobulin Fab Fragments - analysis; Magnesium - chemistry; Methods. Procedures. Technologies; Others; Proteins; Recombinant Proteins - chemistry; Various methods and equipments; Proteins; Immobilized metal-ion affinity chromatography; Purification; Escherichia coli; Organic ligand; Monoclonal antibody; Fab-Fragment; EDTA; immobilized metal affinity chromatography; Affinity adsorbent; Separation method; Affinity chromatography; Bacteria; Magnesium Complexes; Recombinant protein; Enterobacteriaceae; Modified material
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(02)01429-2

Undesired adsorption of host cell proteins poses a big challenge for immobilized metal-ion affinity chromatography (IMAC) purification. In this study, by using His 6-tagged protein Fab OPG C11 from Escherichia coli fermentation as a model, we found that the presence of low concentrations of EDTA–Mg 2+ in feed streams weakens the adsorption but makes it more specific towards polyhistidine tag. By combining EDTA–Mg 2+ treatment and periplasmic extraction, we developed a one-step purification procedure for His 6-tagged recombinant Fab OPG C11 using Ni-IDA (iminodiacetic acid) chromatography. This procedure eliminated the buffer exchange step after periplasmic extraction, which is usually required before IMAC in order to remove EDTA. In addition to savings on time and cost, this procedure eliminates undesired adsorption of most host cell proteins thus significantly improves the purity of polyhistidine-tagged recombinant proteins. The strategy of EDTA–Mg 2+ treatment may have general application potentials.