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Protein engineering of an IgG-binding domain allows milder elution conditions during affinity chromatography
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0001-8993-048X
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0003-0605-8417
2000 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 76, no 03-feb, 233-244 p.Article in journal (Refereed) Published
Abstract [en]

One of the problems in the recovery of antibodies by affinity chromatography is the low pH, which is normally essential to elute the bound material from the column. Here, we have addressed this problem by constructing destabilized mutants of a domain analogue (domain Z) from an IgG-binding bacterial receptor, protein A. In ol-der to destabilize the IgG-binding domain, two protein engineered variants were constructed using site-directed mutagenesis of the second loop of this antiparallel three-helix bundle domain. In the first mutant (Z6C), the second loop was extended with six glycines in order to evaluate the significance of the loop length. In the second mutant (ZL4G), the original loop sequence was exchanged for glycines in order to evaluate the importance of the loop forming residues. Both mutated variants have a lower a-helical content, as well as a lower thermal and chemical stability compared to the parent 2-molecule. The affinity to IgG was slightly lowered in both cases, mainly due to higher dissociation rates. Interestingly, the elution studies showed that most of the bound IgG-molecules could be eluted at a pH as high as 4.5 from columns with the engineered ligands, while only 70% of the bound IgG could be eluted from the matrix with the parent Z as ligand.

Place, publisher, year, edition, pages
2000. Vol. 76, no 03-feb, 233-244 p.
Keyword [en]
affinity chromatography, immunoglobulin G, protein engineering, protein Z, staphylococcal protein A, 4-helix-bundle protein, rop, simulations, resolution, mutations, stability
URN: urn:nbn:se:kth:diva-19497ISI: 000084886400013OAI: diva2:338189
QC 20100525Available from: 2010-08-10 Created: 2010-08-10Bibliographically approved

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