Moreover, theoretically structural studies comparing the native and recombinant Pg-AMP1 forms were also carried out to shed some light on structure–function relationship. Gram-negative bacteria Escherichia coli (ATCC 35218, ATCC 11229), Pseudomonas aeruginosa (ATCC INK 128 mouse 27853), Klebsiella pneumonia (ATCC 13866) and Salmonella typhimurium (ATCC 14028) and Gram-positive bacteria Staphylococcus aureus (ATCC 29213, ATCC 25923), S. aureus MecA (ATCC 33591), Staphylococcus

epidermides (ATCC 12228) were utilized in this report. Bacteria were cultured in Tryptone Soy Broth (TSB-Tryptone 5 g L−1, yeast extract 2.5 g L−1, Dextrose 1 g L−1 and sodium chloride 10 g L−1). The induced E. coli bacteria (BL21-DE3) were cultured in Luria–Bertani broth medium (LB). The gene encoding Pg-AMP1, 168 bp long, was designed to be expressed carrying a His6 tag fused to C-terminal. The codon was optimized

for E. coli expression and the cassette expression was synthesized by Epoch Biolabs and cloned into SmaI site of pBluescriptIISK(−). The expression cassette is composed of Pg-AMP1 gene under control of T7/lac promoter/terminator plus met codon His6 tag encoding a peptide with 62 amino acid residues ( Fig. 1). Recombinant plasmid pBSKPg-AMP1 was used for transformation www.selleckchem.com/products/nutlin-3a.html of E. coli BL21 (DE3) electrocompetent cells (Invitrogen, Carlsbad, CA). The induction was done according to the instruction manual His Trap FF crude (GE, Upsala), using IPTG as an inducer and ampicillin (100 μg mL−1) as select agent. The IPTG induction (0, 0.5 and 1 mM) was

done during 2, 4 or 6 h. Soluble and insoluble fractions were evaluated in each treatment. BL21 (DE3) cells were grown for 4 h from 500 mL of LB at 300 rpm. Pellet cells were obtained from 4500 ×  g at 4 °C after 15 min centrifugation. Pellets were resuspended in lysis buffer (1:10 v/v) containing 50 mM sodium phosphate (pH 7.8), 300 mM sodium chloride, 50 mM potassium chloride, 10% glycerol, 0.5% Triton X-100 and 10 mM imidazole. Enzymatic lysis was performed for 30 min at room temperature with 0.2 mg mL−1 lysozyme, 20 μg mL−1 DNAse, 1 mM MgCl and 1 mM phenylmethylsulfonylfluoride. Mechanical lysis was carried out by sonication on ice for approximately 10 min (in several short bursts). Suspension cells were disrupted Astemizole by sonication (Sonics – Vibra Cell) 20 kHz 100% using the v188 probe on ice four times for 20 s separated by 1 min elapsed time. The suspension was centrifuged at 4500 × g at 4 °C for 30 min. Supernatant carrying soluble proteins were stored −20 °C for subsequent analysis. For each gram of pellet, 3 mL of lysis buffer containing 300 mM sodium chloride, 50 mM sodium phosphate (pH 7.4), 10 mM β-mercaptoethanol and 10 μg mL−1 protease cocktail inhibitor (SIGMA) was added in order to resuspend insoluble fraction. The suspension was kept at room temperature for 30 min and sonicated again for 3× 20 s separated by 1 min interval on ice.