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Competitive elution of protein A fusion proteins allows specific recovery under mild conditions.
KTH, School of Biotechnology (BIO), Proteomics.
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1994 (English)In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 224, no 1, 103-8 p.Article in journal (Refereed) Published
Abstract [en]

A novel system is described for mild elution of fusion proteins by competitive elution. The approach is based on displacement of immobilized fusions containing a monovalent IgG-binding staphylococcal protein A fragment (Z) from an IgG-affinity matrix by a divalent fragment fused to a serum-albumin-binding region derived from streptococcal protein G. Using real-time interaction analysis, the binding (K(aff)) to polyclonal human IgG was found to be 3.3 (+/- 0.4) x 10(8) M-1 for divalent ZZ and 2.0 (+/- 0.1) x 10(7) M-1 for monovalent Z. This more than tenfold difference in binding strength ensures a high efficiency in the elution step. The competitor protein can specifically be removed and recovered from the elution mixture by subsequent passage through a human serum albumin(HSA)-affinity column, leaving only the target fusion protein in the flow-through fraction. Here, we show that a recombinant Klenow fragment of DNA polymerase I expressed in Escherichia coli can be recovered with high yield, and retained activity, from a crude bacterial lysate by IgG-affinity chromatography using mild conditions during both binding and elution.

Place, publisher, year, edition, pages
1994. Vol. 224, no 1, 103-8 p.
National Category
Medical Biotechnology
URN: urn:nbn:se:kth:diva-83833PubMedID: 8076629OAI: diva2:499020
NR 20140805Available from: 2012-02-13 Created: 2012-02-13 Last updated: 2012-02-13Bibliographically approved

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