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Protein purification and crystallization

SB Soledad Baños-Mateos
AR Anne-Marie M. van Roon
UL Ulla F. Lang
SM Sarah L. Maslen
JS J. Mark Skehel
ML Meindert H. Lamers
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WT and mutants M. tuberculosis DnaE1 as well as the short version of DnaE1 (residues 1–941) were expressed in Mycobacterium smegmatis mc21457 cells, kindly provided by W. Jacobs Jr (Albert Einstein College of Medicine). The proteins were purified using nickel-affinity, anion-exchange, and gel filtration columns, and His tags were cleaved with HRV 3C protease. All purification steps were carried out in 50 mM HEPES, pH 7.5, 0.1–1 M NaCl and 2 mM DTT, and stored at −80 °C in 50 mM HEPES pH 7.5, 150 mM NaCl, and 2 mM DTT. DnaE1 crystals were obtained by hanging drop vapor diffusion method from protein at 5 mg/ml and mother liquor solution containing 1.2 M Li2SO4, 0.1 M HEPES pH 7.5. Crystals were transferred to well solution including 25% glycerol before flash freezing in liquid nitrogen.

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