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


Büssow,  Konrad
Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society;

Nyarsik,  Lajos
Max Planck Society;

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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.

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.