The QuikChange kit (Stratagene, La Jolla, CA). Oligonucleotides were obtained from
The QuikChange kit (Stratagene, La Jolla, CA). Oligonucleotides had been obtained from Integrated DNA Technologies (Coralville, IA). The following may be the list of primers utilized to introduce mutations (underlined) into pTP123 KPC-2: P104R: CAAAAATGCGCTGGTTCGCTGGTCACCCATCTC P104L: CAAAAATGCGCTGGTTCTGTGGTCACCCATCTC V240A: CGGAACCTGCGGAGCGTATGGCACGGCAAATG V240G: CGGAACCTGCGGAGGGTATGGCACGGCAAATG H274Y: CAAGGATGACAAGTACAGCGAGGCCGTCATC M49I: CGGTGTGTACGCGATAGATACCGGCTCAGMinimum inhibitory concentration (MIC) determinationsMinimum inhibitory concentrations (MIC’s) for E. coli strain RB791 containing the KPC mutants was determined for imipenem, meropenem and ceftazidime working with Etest strips (Ab Biodisk, Sweden) as outlined by the producers suggestions. The MIC’s of your variants for ampicillin were determined using the broth dilution system in a 100-well microtiter format. Overnight cultures on the variants had been diluted into wells containing two-fold dilutions of ampicillin in a total volume of 300 l LB broth. The plate was allowed to incubate overnight at 37 with continuous shaking and scored for visible growth to identify the MIC.Protein purificationThe relevant blaKPC variant gene in plasmid pTP123 was transformed into E. coli RB791 cells and colonies had been chosen on LB agar containing 12.5 g/mL chloramphenicol. A single colony was applied to inoculate 20 mL LB containing 12.5 g/mL chloramphenicol and permitted to growPLOS Pathogens | DOI:10.1371/journal.ppat.1004949 June 1,15 /Evolution of KPC Carbapenemase Enzymes with Expanded Substrate Profileovernight at 37 . The overnight culture was added to 1 L LB broth containing 12.five g/mL chloramphenicol at a final dilution of 1:100 and subsequently permitted to grow to OD600 0.7 at 37 . Protein expression was induced by addition of 1 M IPTG to a final concentration of 0.2 mM and also the cultures had been grown at 23 overnight. The cells were harvested by PENK Protein Formulation centrifugation at 4000 x g for 20 minutes as well as the pellet frozen for no less than 1 hour at -80 . To release the periplasmic contents, the pellet was resuspended in 50 mL of 10 mM Tris-HCl buffer, pH 8.0 containing 1 tablet of Total Protease Inhibitor ENA-78/CXCL5 Protein Storage & Stability Cocktail (Roche Diagnostics Corporation, Indianapolis, IN) and incubated on ice for 1 hour. Subsequently, osmotic shock was initiated by addition of 50 mL of cold, sterile water. The insoluble material was pelleted by centrifugation at 10,000 g for 1 hour. The supernatant was filtered and passed by way of a HiTrap SP column (GE Healthcare, Piscataway, NJ). The P104R, P104R:V240G and P104R:H274Y mutants bound the column at pH eight.0 and had been eluted making use of a NaCl gradient. The remaining enzyme variants were bound to the column by adjusting the buffer to pH five.five employing MES acid and subsequently they were eluted using a NaCl gradient. The purity of the -lactamase containing fractions was determined applying SDS-PAGE and the pooled fractions have been concentrated and subjected to size exclusion chromatography applying a HiLoad Superdex 75 column (GE Healthcare, Piscataway, NJ). Protein concentrations were determined by measuring the optical density at 280 nm and making use of the following extinction coefficients for respective proteins: 39,545 M-1cm-1 was made use of for KPC2, KPC-4, KPC-6, KPC-11; 41,035 M-1cm-1 for KPC-3, KPC-7, KPC-8, KPC-9, KPC-10 and 39,420 M-1cm-1 for KPC-5. Each of the extinction coefficients were calculated using the `ProtParam’ tool from the Swiss Institute of Bioinformatics on line resource portal [48].Enzyme kineticsMichaelis-Ment.