Purification of overexpressed hexahistidine-tagged BLM N431 as oligomeric complexes Journal Article
| Authors: | Beresten, S. F.; Stan, R.; Van Brabant, A. J.; Ye, T.; Naureckiene, S.; Ellis, N. A. |
| Article Title: | Purification of overexpressed hexahistidine-tagged BLM N431 as oligomeric complexes |
| Abstract: | BLM is a DNA helicase encoded by a gene which is mutated in persons with Bloom's syndrome. The protein is a member of the RecQ subfamily of helicases and contains a central domain constituted by the seven motifs conserved in all helicases. In contrast, the N-terminal portion of BLM lacks similarity to any other known proteins or motifs. We have expressed the first 431 amino acids of this domain as a fusion to a hexahistidine tag (BLM N431) in Escherichia coli. A method of purification was developed which involves elution from Ni-NTA resin in imidazole and EDTA, followed by treatment with DTT and gel filtration on Sephacryl-300. The treatment with EDTA and DTT prevents and disrupts aggregation of BLM N431. The purified protein appears to form hexamers and dodecamers, suggesting that the N-terminal domain of BLM is involved in the organization of the quaternary structure of BLM. |
| Keywords: | animals; gene expression; protein purification; escherichia coli; helicase; protein structure; oligopeptides; adenosine triphosphatases; organometallic compounds; nickel; chelating agents; dna helicases; histidine; amino acid motifs; chromatography, gel; solubility; chromatography, affinity; molecular probes; edetic acid; gel filtration; bloom syndrome; nitrilotriacetic acid; humans |
| Journal Title: | Protein Expression and Purification |
| Volume: | 17 |
| Issue: | 2 |
| ISSN: | 1046-5928 |
| Publisher: | Academic Press Inc., Elsevier Science |
| Date Published: | 1999-11-01 |
| Start Page: | 239 |
| End Page: | 248 |
| Language: | English |
| DOI: | 10.1006/prep.1999.1135 |
| PUBMED: | 10545272 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Article -- Export Date: 16 August 2016 -- Source: Scopus |
Altmetric
Citation Impact
BMJ Impact Analytics
MSK Authors
Related MSK Work