The copper-responsive repressor CopR of Lactococcus lactis is a 'winged helix' protein.

Residue numbering was based on the uncleaved 99 residue CopR-NTD, i.e. M1,S2
CopR-NTD with the his tag was a protein of 99 amino acids, consisting of the 25 amino acid tag, MSYYHHHHHHDYDIPTTENLYFQGA, followed by the N-terminal 74 residues of CopR.

transcription regulator [Lactococcus lactis subsp. lactis IO-1]

predicted transcriptional regulator [Lactococcus lactis subsp. lactis]

transcriptional regulator [Lactococcus lactis subsp. cremoris MG1363]

Transcriptional regulator [Lactococcus lactis subsp. lactis A12]

Negative transcriptional regulator-copper transport operon [Lactococcus lactis subsp. lactis Dephy 1]

transcriptional regulator [Lactococcus lactis subsp. lactis Il1403]

Predicted transcriptional regulator [Lactococcus lactis subsp. cremoris SK11]

Copper transport repressor, CopY/TcrY family [Lactococcus lactis subsp. lactis KF147]

transcriptional regulator [Lactococcus lactis subsp. cremoris NZ9000]

copper transport repressor, CopY/TcrY family [Lactococcus lactis subsp. lactis CV56]

transcriptional regulator [Lactococcus lactis]

For biochemical experiments, E. coli BL21(DE3) (Stratagene) containing plasmid pLlcopyLT298 was grown aerobically at 37 C to an OD550 of 0.4. After 4 h of induction with 1 mM isopropyl-b-D-galactopyranoside (IPTG), the cells were collected by centrifugation for 10 min at 5000 g. The cell pellet was washed twice with 200 ml TG buffer (50 mM Tris-SO4, pH 7.8, 5 % (v/v) glycerol) and resuspended in 5 ml of TG buffer/g of wet cells. The cells were broken by 3 passages through a French press at 40 MPa. The cell debris was collected by centrifugation for 1 h at 90 000 g and the supernatant applied to a Ni-NTA Superflow (Qiagen) column. Following washing of the column with 20 mM imidazole, CopR was eluted with TG buffer containing 200 mM imidazole. Final purification was achieved by gel filtration on a TSK3000G column in TG buffer.

Case, Darden, Cheatham, III, Simmerling, Wang, Duke, Luo, ... and Kollman