Table 1. The source of the ardB and klcA genes investigated Identiﬁer in NCBI protein database Gene Protein Our annotation name Organism NP_752219 ACB01415 AAC75691 YP_122202 BAF33451 NP_990889 ORF14 ArdB yafX ArdB yfjX ArdB plpp0047 ArdB KlcA KlcA pEST4011_14 KlcA ArdBCFT E. coli CFT073 157 ArdBYAF E. coli K12/CP4-6 prophage 152 ArdBYFJ E. coli K12/CP4-57 prophage 152 ArdBLPP L. pneumophila str. Paris 134 KlcA136 B. pertussis BP136 142 KlcAADE A. denitriﬁcans 144 17 832.1 17 419.5 17 303.3 15 409.5 15 659.6 16 026.8 34 410 37 410 37 410 26 740 22 900 21 620 4.83 4.76 4.64 5.05 4.84 4.74 37 37 37 25 30 30 Nucleic Acids Research, 2010, Vol. 38, No. 5 1725 Amino Molecular Molar Predicted Optimal acid weight extinction isoelectric protein length including coeﬃcient point expression N-terminal M 1 cm 1 temperature methionine ( C) Reference (23) (21) (21) (24) (19) (25) The synthesised genes were then ligated into pET24a (kanamycin resistant) at the NdeI and HindIII sites of the expression vector and the anticipated sequence was veriﬁed by DNA sequencing on both strands. Two further ardB genes from E. coli DH5a (yjfX and yafX), Table 1, were PCR-ampliﬁed from genomic DNA and ligated into pET20b (Novagen, Madison, WI, USA). The primers used to amplify yfjX were: forward primer, 50-ACGGTGTCATATGACAACACAGACACAGT-30; reverse primer, 50-GCTAGAATTCTTAATCAATAATG CGCAT-30. The primers used to amplify yafX were: forward primer 50-ACGGTGTCATATGACAACACAG ACGCAGC-30; reverse primer, 50-GCTAGAATTCTCA GTCGATAATACGCAG-30. The NdeI and EcoRI recog- nition sites used in the subsequent ligation are underlined. In both cases an ampliﬁed product of the expected size was obtained. The PCR products were then ligated into the NdeI and EcoRI sites of pET20b (ampicillin resistant). The sequence of the insert was veriﬁed to ensure no mistakes had been introduced during ampliﬁcation. For assessing the eﬀect of expressing klcA136 in NM1041(DE3) (hsd+ clpX ) on growth in the presence and absence of 2-AP, klcA136 was ligated into an expres- sion vector with a carbenicillin selection marker (pET20b). This was achieved by digesting the KlcA136 pET24a expression construct with NdeI and HindIII and ligating the insert into the corresponding sites of pET20b to produce pET20b-klcA136. The extinction coeﬃcients of the proteins were calculated using the method of Gill and von Hippel (31), Table 1. All concentrations quoted for the proteins are based on the concentrations of monomers. The monomer molecular weights of the proteins are shown in Table 1 and include the N-terminal methionine. Expression and puriﬁcation of proteins Escherichia coli BL21(DE3) cells were transformed with the recombinant plasmid and transformants selected using LB agar supplemented with 25 mg/ml kanamycin or 100 mg/ml carbenicillin. A single colony was picked and grown in LB medium plus 25 mg/ml kanamycin or 100 mg/ml carbenicillin at 37 C to an optical density at 600 nm (OD600) of 0.5. Heterologous gene expression was induced by the addition of IPTG to 1 mM and the growth continued for a further 3 h at the temperature given in Table 1 before harvesting. For four of the chosen genes, a prominent protein band corresponding to the predicted protein mass was visible on SDS– polyacrylamide gels of induced cells of BL21(DE3) con- taining the recombinant plasmid that was not visible in the negative control of the vector alone in BL21(DE3). The A. denitriﬁcans klcA and L. pneumophila ardB could not be expressed at levels suﬃcient for puriﬁcation. For protein production, a single colony of was used to inocu- late 100 ml of LB medium supplemented with 25 mg/ml kanamycin or 100 mg/ml carbenicillin. The starter culture was grown to an OD600 of 0.5–0.8 at 37 C and used to inoculate 10 L of LB containing 25 mg/ml kanamycin or 100 mg/ml carbenicillin and the cells grown at 37 C to an OD600 of 0.5. Heterologous gene expression was induced as described above. Typically 50 g of cells were obtained from 10 l of culture. Cells were harvested by centrifugation (6000g, 4 C, 15 min) and the cell pellet was stored at 20 C until required. Approximately 20 g of cell pellet was defrosted on ice for 30 min and resuspended in 200 ml buﬀer A (20 mM Tris–HCl pH 8.0, 7 mM 2-mercaptoethanol). A cocktail of protease inhibitors (Complete Protease Inhibitor Cocktail Tablet; Roche) was added to the cell suspension (1 tablet per 50 ml solution). The cells were disrupted by sonication on ice using a Soniprep 150 sonicator (Sanyo, Tokyo, Japan) ﬁtted with a 9 mm diameter probe (5 1 min bursts with 15 s interval between each burst). The cell lysate was centrifuged at 20 000g for 1 h at 4 C and the supernatant was ﬁltered through a 0.45 mm ﬁlter unit (Sartorius AG, Goettingen, Germany). The clariﬁed extract was then loaded onto a 30 cm 1 cm diameter DEAE anion exchange column equilibrated with buﬀer A, at a ﬂow rate of 60 ml/h. The column was washed with 3 column volumes of buﬀer A to remove unbound material. Bound proteins were then eluted using a 500 ml gradient of 0–0.7 M NaCl in buﬀer A at a ﬂow rate of 20 ml/h. Fractions that gave a UV absorbance were subsequently analysed by SDS–PAGE and those contain- ing ArdB were pooled and concentrated using a spin concentrator with a 5 kDa MWCO (VivaScience AG, Hannover, Germany) to a ﬁnal volume of 5 ml. The sample was then loaded on a Superdex-200 column (GE Healthcare, Uppsala, Sweden), equilibrated with buﬀer A + 0.2 M NaCl, and the protein was eluted at a ﬂow rate of 10 ml/h. The fractions were subsequently