The forward primer Ef-ccpAU introduced www.selleckchem.com/products/GDC-0941.html a NdeI site around the initiation codon of the ccpA gene, and the backward primer Ef-ccpAL introduced a BamHI site downstream of the stop codon (Table 2 and Table 3). The PCR product was double-digested and ligated into the corresponding restriction sites of vector pET-28a(+) (Novagen). The resulting plasmid, named pET-CcpA, codes for CcpA extended with a 6-histidine tag at the N terminus (Table 2). The correct sequence of the
insert was confirmed, and the plasmid was subsequently introduced into E. coli BL21 (DE3) for ccpA overexpression. E. coli BL21 (DE3) harboring the pET-ccpA plasmid was grown in LB at 37°C until an O.D.600= 0.6 was reached. Next, CcpA
expression was induced by addition of 0.5 mM IPTG. Following an overnight culture, cells were harvested by centrifugation and resuspended in ice-cold Tris-HCl buffer (50 mM, pH 8.0), containing 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 300 mM NaCl and 5% glycerol. Cells were disrupted by passing them through a French Pressure cell. The suspension was centrifuged and the selleckchem supernatant was mixed with nickel-nitrilotriacetic acid agarose (Novagen). His6-CcpA was eluted with imidazole and the purified protein was dialyzed against binding buffer (25 mM Tris-HCl, pH 6.6, 150 mM NaCl and 10% glycerol) and stored at -80 °C for further studies. find more Lactobacillus casei HprK/P(V267F) and Enterococcus
casseliflavus HPr were overproduced using pQE30 vector and purified following a standard protocol, as described previously [42]. Seryl-phosphorylated E. casseliflavus HPr was prepared as described by Mazé et al. [43] using L. casei V267F mutant HprK/P, which possesses kinase activity but has almost completely lost the phosphorylase function [42]. About 0.5 mg of HPr was incubated for 30 min at 37°C in 1 ml final volume containing also 10 μg of HprK/P(V267F), 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM fructose-1,6-bisphosphate (FBP), and 5 mM ATP. To inactivate HprK/P(V267F), the samples were heated Fossariinae for 5 min at 75°C before they were desalted on PD-10 columns (GE Healthcare Life Sciences) to remove ATP and FBP and lyophilized. HPr and P-Ser-HPr were separated by electrophoresis on nondenaturing 12.5% polyacrylamide gels and visualized by staining with Coomassie blue; usually 99% of the HPr was converted into P-Ser-HPr. DNA labeling The synthetic oligonucleotides EfHpromU, Efint4_Lo, EfbsPoadA were labeled at their 5′ ends using [γ-32P]ATP (NEN PerkinElmer). The labeled oligonucleotides were purified using a Zeba Desalt Spin Column (Thermo scientific). DNA fragments containing different cre sites were amplified by PCR; for the amplicons A, B and C we used the primer pairs EfHpromU-EfcitNUp, EfbscitN-Efint4_Lo and EfbsPoadA-Efbsint_Up, respectively.
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