Proteins
DNA sequences of primers and substrates, plasmids and E. colistrains are detailed in Supplementary data. E. coli MG1655 gene
b1653, encoding Lhr, was PCR-amplified from genomic DNA, and cloned into
pET14b using Nde I and Hin DIII restriction sites generating
pRJB28 for expression of hexa-histidine tagged Lhr. Lhr-CTD (amino acids
876-1538) was amplified and cloned by ligation independent cloning (LIC)
The resulting plasmid based on vector pNH-TrxT (GU269914.1 [23]).
These plasmids were used to generate LhrD1536A using
mutagenic primers in PCR by Q5 hot start polymerase, and resulting
reactions were treated with Dpn I, T4 polynucleotide kinase, and
DNA ligase. Plasmid DNA was extracted and sequenced from colonies after
transforming reaction mixtures into E. coli .
Lhr and C-Lhr proteins were over-expressed in E. coli Rosetta 2
cells grown in MU broth with ampicillin and chloramphenicol selection.
Cells were grown with shaking at 37°C to OD600 of 1.2
and transferred to an ice slurry for cooling before addition of IPTG
(0.8 mM). Growth was continued for 10 hours at 18°C, cells were
harvested and resuspended in 20 mM HEPES pH 8.0, 1.5 M ammonium sulfate,
20 mM imidazole, 10% (w/v) glycerol (Ni-NTA buffer A) containing 0.1 mM
phenylmethylsulphonyl fluoride (PMSF). This process and purification was
also followed for obtaining LhrD1536A protein. Cells
were thawed and sonicated on ice, clarified by centrifugation, and
soluble proteins were loaded into a 5 ml butyl sepharose column
equilibrated with 20 mM HEPES pH 8.0, 1.5 M ammonium sulfate, 10% (w/v)
glycerol (hydrophobic salt buffer A). The column was washed with 20 mM
HEPES pH 8.0, 900 mM ammonium sulfate. Then a 5 ml Ni-NTA column
pre-equilibrated with Ni-NTA buffer A was attached in tandem with the
butyl sepharose column and columns washed with 20 mM HEPES pH 8.0 and
10% glycerol until no proteins were detectable by UV monitoring as
eluting from the columns. The butyl sepharose column was removed and Lhr
was eluted from the Ni-NTA column by increasing imidazole to 500 mM in
20 mM HEPEs and 10% glycerol. Lhr-containing fractions were pooled and
dialyzed overnight into 20 mM HEPES pH 8.0, 150 mM NaCl, 10% (w/v)
glycerol (low salt buffer A) and loaded into a 1 ml Q-sepharose column.
Lhr eluted in an increasing gradient of NaCl to 1.5 M. Lhr fractions
were pooled and dialyzed overnight for storing in 20 mM HEPES pH 8.0,
150 mM NaCl and 35% (w/v) glycerol for aliquoting, flash freezing, and
storage at -80°C. E. coli uracil-DNA glycosylase and
formamidopyrimidine DNA glycosylase (Fpg) control proteins (Figure 3)
were purchased from New England Biolabs.