English
 
User Manual Privacy Policy Disclaimer Contact us
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT
  Rapid purification and crystal structure analysis of a small protein carrying two terminal affinity tags

Mueller, U., Büssow, K., Diehl, A., Bartl, F. J., Niesen, F. H., Nyarsik, L., et al. (2003). Rapid purification and crystal structure analysis of a small protein carrying two terminal affinity tags. Journal of Structural and Functional Genomics, 4(4), 217-225. doi:10.1023/B:JSFG.0000016119.50040.a3.

Item is

Basic

show hide
Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-8963-7 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-8964-5
Genre: Journal Article
Alternative Title : J Struct Funct Genomics

Files

show Files

Locators

show

Creators

show
hide
 Creators:
Mueller, Uwe, Author
Büssow, Konrad1, Author              
Diehl, Anne, Author
Bartl, Franz J., Author
Niesen, Frank H., Author
Nyarsik, Lajos2, Author
Heinemann, Udo, Author
Affiliations:
1Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1433550              
2Max Planck Society, ou_persistent13              

Content

show
hide
Free keywords: affinity chromatography; protein purification; SH3 domain; structural genomics; X-ray crystallography
 Abstract: Small peptide tags are often fused to proteins to allow their affinity purification in high-throughput structure analysis schemes. To assess the compatibility of small peptide tags with protein crystallization and to examine if the tags alter the three-dimensional structure, the N-terminus of the chicken agr-spectrin SH3 domain was labeled with a His6 tag and the C-terminus with a StrepII tag. The resulting protein, His6-SH3-StrepII, consists of 83 amino-acid residues, 23 of which originate from the tags. His6-SH3-StrepII is readily purified by dual affinity chromatography, has very similar biophysical characteristics as the untagged protein domain and crystallizes readily from a number of sparse-matrix screen conditions. The crystal structure analysis at 2.3 A resolution proves native-like structure of His6-SH3-StrepII and shows the entire His6 tag and part of the StrepII tag to be disordered in the crystal. Obviously, the fused affinity tags did not interfere with crystallization and structure analysis and did not change the protein structure. From the extreme case of His6-SH3-StrepII, where affinity tags represent 27% of the total fusion protein mass, we extrapolate that protein constructs with N- and C-terminal peptide tags may lend themselves to biophysical and structural investigations in high-throughput regimes.AbbreviationsSH3 domain – Src homology 3 domain; His6-SH3-StrepII – agr-spectrin SH3 domain with N-terminal His6 and C-terminal StrepII affinity tag; GST – glutathione S-transferase; MBP – maltose-binding protein; aa – amino acid(s); rms – root-mean-square; MC – metal-chelating.

Details

show
hide
Language(s): eng - English
 Dates: 2003-12
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Method: -
 Identifiers: eDoc: 230628
DOI: 10.1023/B:JSFG.0000016119.50040.a3
 Degree: -

Event

show

Legal Case

show

Project information

show

Source 1

show
hide
Title: Journal of Structural and Functional Genomics
  Alternative Title : J Struct Funct Genomics
Source Genre: Journal
 Creator(s):
Affiliations:
Publ. Info: -
Pages: - Volume / Issue: 4 (4) Sequence Number: - Start / End Page: 217 - 225 Identifier: ISSN: 1345-711X