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Automated purification of soluble histidine tagged integrase of Tn21 expressed in E. coli cells in low amounts
Microbial Cell Factories volume 5, Article number: P48 (2006)
Deca-Histidine tagged integrase Tn21, a basic DNA-binding protein with a molecular mass of 38 kDa, when expressed in E. coli bacteria resulted in inclusion bodies. To obtain native, biologically active protein, it was decided to purify only cytoplasmic soluble integrase, present in low amounts. The optimal growth conditions, low temperature and 20 hours of growth period were used to cultivate the bacterial cells to increase the amount of the soluble integrase. LC-MS/MS techniques were used as analytical techniques to measure concentration of the expressed protein during growth optimization.
ÄKTAxpress™, an automated system, which allows multi-step purification of protein, was used to purify the enzyme. Additional ready-to-run protocols increased the flexibility of combining different chromatographic techniques (affinity and ion exchange chromatography, gel filtration and desalting) in the desired order.
All purifications were performed at + 6°C as the protein is labile. The method consisted of an immobilized metal chelating chromatography as capture step. A column prepacked with Ni Sepharose™ High Performance was used to capture the deca-histidine tagged integrase. The system allowed the automated collection of peaks in the storage loops and the transfer of the major peak to a gel filtration column (Hi Load™ 16/60 Superdex™ 75 prep grade). The purified protein was analyzed using SDS-PAGE and MS techniques.
Using the optimized experimental conditions, 5 mg of pure labile integron integrase of Tn21 was purified, sufficient enough for functional studies as well as crystallization screening.
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Grigorescu, I., Andersson, A., Galin, M. et al. Automated purification of soluble histidine tagged integrase of Tn21 expressed in E. coli cells in low amounts. Microb Cell Fact 5, P48 (2006). https://doi.org/10.1186/1475-2859-5-S1-P48
- Inclusion Body
- Exchange Chromatography
- Chromatographic Technique
- Optimal Growth Condition