Truncated HydH5 N-terminally 6×His-tagged derivative containing a 161 amino acid LYZ2 domain was obtained by amplification of orf58 with oligonucleotides LYZF (5′- CGGGATCCCAAGATACTTAAAGGCAAGGGGA- 3′) and LYZR (5′- CACACCTCTGAATTCATATTAATCTCTTG- 3′) which generated a www.selleckchem.com/products/AG-014699.html 474 bp PCR fragment flanked by the restriction sites BamHI and EcoRI (as a consequence of the cloning process, 12 additional amino acid residues, Met-Gly-Ser-Ser-His-His-His-His-His-His-Ser-Gln, were introduced at the N-terminal region and 2 amino acid residues, Arg-Asp, at the C-terminal region of the formal 147 amino acid LYZ2 domain defined by the PFAM conserved domain database). Likewise, N-terminal 6×His-tagged CHAP domain was

also obtained with the oligonucleotide pair CHAPF (5′- CGGGATCCCGAAGTAGTAGAGTGGGC- 3′) and CHAPR (5′- GGAATTCTTATCTAACAAAATGTGTTACTC -3′) yielding check details a 424 bp PCR product (12 additional amino acid residues, Met-Gly-Ser-Ser-His-His-His-His-His-His-Ser-Gln, were introduced at the N-terminal region of the formal 135 amino acid CHAP domain). Restricted PCR fragments were cloned into plasmid pETDuet-1 (pETDuet1-LYZ2 and pETDuet1-CHAP), respectively. All DNA cloning steps were initially performed in E. coli DH10B and then electroporated into E. coli BL21(DE3)/pLysS and/or E. coli Rosetta (DE3). The integrity of all the clones

was verified by both restriction enzyme site profiling and DNA sequence analysis. Heterologous overexpression and protein purification High-level expression of the His-tagged protein HydH5 was achieved in E. coli Rosetta (DE3), while the LYZ2 and CHAP HydH5-derived truncations were expressed in E. coli BL21(DE3)/pLysS. Exponentially growing cultures (A600 0.5) were induced with 1 mM IPTG (isopropyl- beta-D-thiogalactopyranoside). After Lazertinib chemical structure incubation for 30 min at 37°C, rifampicin was added

to a final concentration of 240 μg/ml and incubation continued for 4 h. Cells were pelleted, washed with 50 mM phosphate buffer, pH 7, and frozen at -80 °C. The recombinant proteins were not found in the soluble fraction, and were thus purified from inclusion bodies. Cell pellets from 1.2 l cultures were resuspended in 10 ml per g of wet weight of 1× cell resuspension buffer (iFOLD Protein Refolding System P-type ATPase 2) (Novagen, Madison, USA) and sonicated on ice (15×5 s pulses with 15 s recovery between pulses) following the manufacturer’s instructions. Inclusion bodies containing HydH5, LYZ2 and CHAP proteins were obtained via centrifugation (8000 × g) and stored as pellets at -80°C. They were denatured in iFold Guanidine denaturation buffer and optimal conditions for correct folding (highest activity and solubility) were determined with the iFold protein refolding matrix and via antimicrobial assays. The highest activity and solubility was obtained by refolding HydH5 and LYZ2 in buffer A (HEPES 50 mM, NDSB-201 0.5 M, CaCl2 0.