CRP was expressed and purified in a similar manner. Primers were used to amplify crp with the restriction sites HindIII and XhoI on the 5′ and 3′ ends, respectively (Table 2). The 41 bases immediately upstream of crp were included to ensure that the native bacterial translation signals were present. The downstream primer included the last codon of the crp open reading frame, excluding the stop codon, to allow for the fusion of https://www.selleckchem.com/products/prn1371.html a multiple-histidine tag. The PCR product was cloned into pGEM-T and subsequently subcloned into pET-24(+) (Novagen, Madison, WI) using the HindIII and XhoI sites.
The resulting plasmid, pJJ276, was expected to express CRP with a carboxy-terminal His•Tag. Protein expression was induced using the Overnight Express Autoinduction System 1 (Novagen) grown at 37°C overnight. Expressed protein was purified using the BD TALON Metal Affinity Resin (BD Biosciences, Palo Alto, CA). Purification was performed in native conditions following the manufacturer’s protocol and using the suggested
TALON buffers. Eluted fractions were examined by SDS-PAGE and fractions containing CRP were pooled. Protein was concentrated using an selleck chemicals llc Amicon Ultra centrifugation filter and desalted as described above. The protein concentration was determined using the NanoDrop ND-1000 Spectrophotometer and an extinction coefficient of 21,555 M-1 cm-1. Purified protein was stored at 4°C. Electrophoretic mobility shift assay Electrophoretic mobility shift assay (EMSA) was used to study the binding of SiaR and CRP to potential promoter sequences as done previously [14]. The probe for EMSA was amplified by
PCR using primer pairs P146F1 and P146R4 (Table 2), resulting in a probe that spans the region from the nanE MTMR9 start codon to +18 of the siaPT transcript. Binding reactions were prepared using the EMSA Kit (Molecular Probes, Eugene, OR) following the manufacturer’s directions with some modifications. Binding reactions consisted of the binding buffer (150 mM KCl, 0.1 mM DTT, 0.1 mM EDTA, 10 mM Tris, pH 7.4), the DNA probe (15 nM), and 1 μM SiaR and/or CRP. Control reactions without protein were set up for each probe. Reactions were incubated at room temperature for 20 minutes. After incubation, 6× EMSA gel-loading solution was added and reactions were loaded onto a 6% DNA Retardation Gel (Invitrogen) with prechilled 0.5× TBE buffer and run at 200 V for 60 minutes. After electrophoresis, the gel was stained with SYBR Green EMSA gel stain and bands were visualized by UV transillumination. Images were captured using a Kodak EDAS 120 camera with an EDAS 590 mm filter (Eastman Kodak Company, Rochester, NY). cAMP was added to reactions when indicated to a final concentration of 100 μM. Primer extension analysis Primer extension analysis was used to identify the transcriptional start sites for both nan and siaPT operons.