Negatively charged purification tags for selective anion-exchange recovery
2004 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 17, no 11, 779-786 p.Article in journal (Refereed) Published
A novel strategy for the highly selective purification of recombinant fusion proteins using negatively charged protein domains, which were constructed by protein design, is described. A triple alpha-helical domain of 58 amino acids was used as scaffold. Far-ultraviolet circular dichroism measurements showed that the designed domains had very low alpha-helicity in a low-conductivity environment in contrast to the scaffold. The secondary structure could be induced by adding salt, giving a structure comparable to the parental molecule. Further studies showed that the new domains were able to bind to an anion exchanger even at pH values down to 5 and 6. Gene fusions between one of the designed domains and different target proteins, such as green fluorescent protein (GFP), maltose binding protein (MBP) and firefly luciferase, were also constructed. These gene products could be efficiently purified from whole cell lysates at pH 6 using anion-exchange chromatography.
Place, publisher, year, edition, pages
2004. Vol. 17, no 11, 779-786 p.
ion-exchange chromatography, protein A, protein design, Z domain
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:kth:diva-8272DOI: 10.1093/protein/gzh092ISI: 000227133500003ScopusID: 2-s2.0-14644444571OAI: oai:DiVA.org:kth-8272DiVA: diva2:13550
QC 20100923 QC 201109152005-11-022005-11-022011-09-15Bibliographically approved