Skip Navigation


Nucleic Acids Research Advance Access originally published online on December 11, 2008
Nucleic Acids Research 2009 37(2):e15; doi:10.1093/nar/gkn992
This Article
Right arrow Full Text Freely available
Right arrow Print PDF (4676K) Freely available
Right arrow Screen PDF (557K) Freely available
Right arrowOA All Versions of this Article:
37/2/e15    most recent
gkn992v1
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 arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Ederth, J.
Right arrow Articles by Sanyal, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ederth, J.
Right arrow Articles by Sanyal, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, 2009, Vol. 37, No. 2 e15
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

A single-step method for purification of active His-tagged ribosomes from a genetically engineered Escherichia coli

Josefine Ederth, Chandra Sekhar Mandava, Santanu Dasgupta and Suparna Sanyal*

Department of Cell and Molecular Biology, Uppsala University, S-751 24 Uppsala, Sweden

*To whom correspondence should be addressed. Tel: +46 18 471 4220; Fax: +46 18 471 4262; Email: suparna.sanyal{at}icm.uu.se

Received July 10, 2008. Revised November 21, 2008. Accepted November 25, 2008.

With the rapid development of the ribosome field in recent years a quick, simple and high-throughput method for purification of the bacterial ribosome is in demand. We have designed a new strain of Escherichia coli (JE28) by an in-frame fusion of a nucleotide sequence encoding a hexa-histidine affinity tag at the 3'-end of the single copy rplL gene (encoding the ribosomal protein L12) at the chromosomal site of the wild-type strain MG1655. As a result, JE28 produces a homogeneous population of ribosomes (His)6-tagged at the C-termini of all four L12 proteins. Furthermore, we have developed a single-step, high-throughput method for purification of tetra-(His)6-tagged 70S ribosomes from this strain using affinity chromatography. These ribosomes, when compared with the conventionally purified ones in sucrose gradient centrifugation, 2D-gel, dipeptide formation and a full-length protein synthesis assay showed higher yield and activity. We further describe how this method can be adapted for purification of ribosomal subunits and mutant ribosomes. These methodologies could, in principle, also be used to purify any functional multimeric complex from the bacterial cell.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.


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.