The inserted fragments were amplified by PCR and complete fragmen

The inserted fragments were amplified by PCR and complete fragment sequences were determined using a 3130 Genetic Analyzer (Applied Biosystems). The S. marcescens nucleotide sequences determined in this work have been deposited in the DDBJ/EMBL/GenBank databases under the following accession numbers: AB505202

and AB505203 for the phlA and phlB genes of S. marcescens niid 298. Construction of mutant strains A one-step inactivation method [19–21] was used to obtain shlBA and phlAB deletion mutants. For construction of a shlBA deletion mutant, PCR products were amplified from pKD4 [19] with primers ShlBA5′ (5′-GGTTAACCTCATGGATTGGGCTGGCTGCCCCGGCGGCCTCTCATAGTGTAGGCTGGAGCTGCTTC-3′) PARP inhibitor and ShlBA3′ (5′-GCAAAACTCCACGCCTGCCGTCATGCTTCATGTCACTGTCAGCAACATATGAATATCCTCCTTAGT-3′), which flank the 5′ and 3′ termini of the shlBA gene with 45 bp homology, and electroporated into S. marcescens niid THZ1 order 298 carrying pKD46 [20]. For construction of a phlAB deletion mutant, primers PhlAB5′ (5′-AGCGCCAGTAAGGCTATCGCCAGCGCCCGCCGCAAGCGACCCCCTCATATGAATATCCTCCTTAGT-3′) and PhlAB3′ (5′-TGCCTAAGAAAAAACCGCCTGTACAGGCGGTTTTTTTATGGGCGTCATATGAATATCCTCCTTAGT-3′) were used. The correct mutation was verified by PCR using three different primer sets as described previously [20]. Preparation of purified PhlA The full-length phlA gene was obtained from S. marcescens niid 298 genomic DNA by PCR with primers phlA5′

(5′-GAATTCCATATGAGTATGCCTTTAAGTTTTACCTCTG-3′) and phlA3′ (5′-GCTATCTAGATCAGGCATTGGCCTTCGCCTC-3′). The 5′- and 3′-termini of the PCR product were NdeI and XbaI restriction enzyme

sites, respectively. The PCR fragment cleaved by these restriction enzymes was inserted Endonuclease into NdeI/XbaI-digested pCold1, which has a histidine tag site at the 5′-terminus, and used to transform E. coli DH5α. The resulting plasmid, pCold1-phlA, was introduced into E. coli BL21 harboring pG-KJE8 [22]. The transformant was used for expression of His-PhlA according to the manufacturer’s instructions. To purify the His-PhlA recombinant protein, cells were harvested, lysed by lysis buffer [50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, and protease inhibitors (one tablet of inhibitor mixture (Complete, Roche)/25 ml)], incubated in a final concentration of 1 mg lysozyme/ml for 20 min on ice, and then disrupted by sonication. Cell debris was removed by centrifugation. The supernatant was analyzed by affinity chromatography using Ni-NTA agarose (Qiagen) under native conditions without a protease inhibitor. After dialysis against PBS, the purified selleckchem protein was concentrated by Amicon Ultra-15 (MWCO = 30 K; Millipore). The protein concentration was determined using a BCA Protein Assay Kit (Pierce). We obtained approximately 1.9 mg purified recombinant protein (His-PhlA) from one liter of culture. The purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie blue staining.

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