Proteins Sample Clauses

Proteins. The UVDE gene fragment encoding residues 229 - 599 was amplified by PCR and cloned into the BamHI and BstEII restriction sites of the expression vector pET16b (Novagen), generating an N-terminal fusion of the truncated gene to a 10 X His tag. The resulting plasmid (pETUVDE∆228) was introduced into E. coli BL21/codon+ cells (Studier et al., 1990) and the Δ228-UVDE protein purified from a 2 l culture 3 h after induction by IPTG. The cells were lysed by sonication in 6 ml lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10 mM β-mercaptoethanol, 10 % glycerol, 1 % Triton X-100) and separated into a soluble and an insoluble fraction by centrifugation at 37,000 rpm for 30 minutes. The supernatant was loaded on a HiTrap-chelating Ni column, equilibrated with 20 mM Tris pH 7.5 containing 20 mM imidazole and the protein was eluted with a gradient of 20 mM to 250 mM imidazole in the same buffer. Pooled fractions of Δ228-UVDE were loaded on a hydroxyapatite column, which was equilibrated with 10 mM KPO4 (pH 6.5). Subsequently, the protein was eluted using a gradient from 200 mM to 400 mM KPO4 (pH 6.5). For further purification, the UVDE fractions were applied to a P11 phosphocellulose column, equilibrated with 300 mM KPO4 (pH 6.5) and eluted with 1 M NaCl. Finally, the UVDE containing fractions were applied to a Nap5 gel filtration column and eluted with 20 mM Tris pH 7.5, 150 mM NaCl, 10 % glycerol. The UVDE containing fractions showed more than 95 % purity. The Y358A point mutation was constructed by PCR and verified by sequencing. The mutant UVDE protein was purified using the same purification procedure as described for the wild type enzyme and showed the same elution/purification profile. The Endonuclease IV (Endo IV) and Endonuclease III (Endo III) enzymes were obtained commercially (New England Biolabs). DNA substrates The 30 bp DNA substrates used in this study are summarised in Table 1. The oligonucleotides containing CPD or (6-4)PP lesions were synthesised as described (Iwai, 2006). The 30 bp substrates containing an abasic site (via incorporation of a tetrahydrofuran-dSpacer), thymine glycol (TG) or 2-AP were obtained commercially (Eurogentec, Belgium). The DNA substrates were 5’ radioactively labelled using polynucleotide kinase as described (Xxxxxxxxx et al., 2002). For 3’ labelling the damaged top strands were annealed to a corresponding bottom strand with one additional G residue at its 5’ end. Subsequently the DNA fragments were incubated with Klenow polymerase a...
Sample 1
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Related to Proteins

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