Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow FREE Full Text (PDF) Freely available
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 ISI Web of Science
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 arrow Search for citing articles in:
ISI Web of Science (14)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Gräslund, T.
Right arrow Articles by Hober, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gräslund, T.
Right arrow Articles by Hober, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Protein Engineering, Vol. 13, No. 10, 703-709, October 2000
© 2000 Oxford University Press

Charge engineering of a protein domain to allow efficient ion-exchange recovery

Torbjörn Gräslund1, Gunnel Lundin2, Mathias Uhlén1, Per-Åke Nygren1 and Sophia Hober1,3

1 Department of Biotechnology, Royal Institute of Technology (KTH), S-100 44 Stockholm and 2 Department of Genetics, Stockholm University, S-106 91 Stockholm, Sweden

We have created protein domains with extreme surface charge. These mutated domains allow for ion-exchange chromatography under conditions favourable for selective and efficient capture, using Escherichia coli as a host organism. The staphylococcal protein A-derived domain Z (Zwt) was used as a scaffold when constructing two mutants, Zbasic1 and Zbasic2, with high positive surface charge. Far-ultraviolet circular dichroism measurements showed that they have a secondary structure content comparable to the parental molecule Zwt. Although melting temperatures (Tm) of the engineered domains were lower than that of the wild-type Z domain, both mutants could be produced successfully as intracellular full-length products in E.coli and purified to homogeneity by ion-exchange chromatography. Further studies performed on Zbasic1 and Zbasic2 showed that they were able to bind to a cation exchanger even at pH values in the 9 to 11 range. A gene fusion between Zbasic2 and the acidic human serum albumin binding domain (ABD), derived from streptococcal protein G, was also constructed. The gene product Zbasic2–ABD could be purified using cation-exchange chromatography from a whole cell lysate to more than 90% purity.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Protein Eng Des SelHome page
M. Hedhammar, T. Graslund, M. Uhlen, and S. Hober
Negatively charged purification tags for selective anion-exchange recovery
Protein Eng. Des. Sel., November 1, 2004; 17(11): 779 - 786.
[Abstract] [Full Text] [PDF]

Home page
 Protein Eng Des SelHome page
M. Wikman, A.-C. Steffen, E. Gunneriusson, V. Tolmachev, G.P. Adams, J. Carlsson, and S. Stahl
Selection and characterization of HER2/neu-binding affibody ligands
Protein Eng. Des. Sel., May 1, 2004; 17(5): 455 - 462.
[Abstract] [Full Text] [PDF]

Home page
 Protein Eng Des SelHome page
S. Gulich, M. Linhult, S. Stahl, and S. Hober
Engineering streptococcal protein G for increased alkaline stability
Protein Eng. Des. Sel., October 1, 2002; 15(10): 835 - 842.
[Abstract] [Full Text] [PDF]


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.