Skip Navigation



PEDS Advance Access published online on December 3, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh092
This Article
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
17/11/779    most recent
gzh092v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Hedhammar, M.
Right arrow Articles by Hober, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hedhammar, M.
Right arrow Articles by Hober, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Protein Engineering, Design & Selection © Oxford University Press 2004; all rights reserved
Received July 6, 2004
Revised November 1, 2004
Accepted November 22, 2004

Article

Negatively charged purification tags for selective anion-exchange recovery

M. Hedhammar 1, T. Gräslund 1, M. Uhlén 1, and S. Hober 1*

1 Dept. of Biotechnology, Royal Inst. of Technology (KTH), SE-106 91 Stockholm, Sweden

* To whom correspondence should be addressed.
S. Hober, E-mail: sophia.hober{at}biotech.kth.se


  Abstract

Here we describe a novel strategy for highly selective purification of recombinant fusion protein using highly negatively charged protein domains which were constructed by protein design. 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 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.

Keywords: ion-exchange chromatography; protein design; Protein A; Z domain.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.