Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner
2002 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 942, no 1-2, 157-166 p.Article in journal (Refereed) Published
To achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography, a novel basic protein domain is used as a purification handle. The proteolytic instability usually encountered for basic peptide tags is avoided by the use of a highly constrained α-helical domain based on staphylococcal protein A into which positively charged amino acids have been introduced. Here we show that this domain, consisting of 58 amino acids with a calculated isoelectric point (pI) of 10.5, can be used to efficiently capture different fused target proteins, such as a bacterial DNA polymerase (Klenow fragment), a viral protease (3C) and a fungal lipase (Cutinase). In contrast to standard cation-exchange chromatography, efficient capture can be achieved also at a pH value higher than the pI of the fusion protein, demonstrated here by Zbasic-Klenow polymerase (pI≈5.8) and ZZ-Cutinase-Zbasic (pI≈7.2) both purified at a pH of 7.5. These results show that the Zbasic domain is able to confer a regional concentration of positive charge on the fusion protein even at a relatively high pH. Hence, the data suggest that this domain could be used for highly efficient and selective capture of target proteins at conditions where most host-cell proteins do not bind to the chromatographic resin. The obtained purity after this one-step procedure suggests that the strategy could be an alternative to standard affinity chromatography. Methods for site-specific proteolysis of the fusion proteins to release native target proteins are also discussed.
Place, publisher, year, edition, pages
2002. Vol. 942, no 1-2, 157-166 p.
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:kth:diva-8270DOI: 10.1016/S0021-9673(01)01413-3ISI: 000173120900015OAI: oai:DiVA.org:kth-8270DiVA: diva2:13548
QC 201006092005-11-022005-11-022012-02-13Bibliographically approved