Histrap hp crude columns

The strains and plasmids used to clone the potential GR gene are presented in Table 1, and the primers are presented in Table 2. To express and purify the recombinant protein of F0726_RS04210, the coding sequence was amplified using primers F-gr and R-gr. The fragment was digested by NdeI and XhoI and inserted into NdeI-XhoI-treated pET28a, to generate recombinant plasmid pET28a-gr. Successful insertion of the coding sequences of target genes was confirmed by sequencing; then, the recombinant plasmids were transformed into E. coli BL21(DE3) cells. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the final concentration of 0.4 mM to induce the expression of recombinant protein at 25°C for at least 5 h. Recombinant proteins were analyzed using 10% SDS-PAGE and purified using HisTrap™ HP Crude Columns (GE Health) and AmiconUltra-15 Centrifugal Filter Units with Ultracel-3 membranes (Merck Millipore). Finally, the protein concentrations were determined using a Pierce^®BCA Protein Assay Kit.

The coding sequences of A5904_0421, A5904_0790 and A5904_1112 were amplified from A. caldus MTH-04 genomic DNA using PrimeSTAR^® HS DNA Polymerase (TaKaRa) with the primer pairs 0421orfF/0421orfR, 0790orfF/0790orfR and 1112orfF/1112orfR (S2 Table), respectively. The fragments were digested with NdeI-XhoI and ligated into NdeI-XhoI treated pET22b(+) to generate the recombinant plasmids pET22b-0421, pET22b-0790 and pET22b-1112. Correct clones were confirmed by sequencing, then the plasmids were transformed into E. coli BL21(DE3) cells. Expression of recombinant proteins was induced by the addition of 0.4 mM IPTG (isopropyl-β-D-thiogalactopyranoside) at 25°C for 5 h. Cells were collected by centrifugation and washed twice with ice-cold 20 mM NaH₂PO₄ buffer (pH 7.4) containing 30 mM imidazole and 500 mM NaCl, and lysed by sonication at 4°C. Supernatants were collected by centrifugation at 13,400×g for 30 min at 4°C and the recombinant proteins were analyzed by 10% (wt/vol) SDS-PAGE gels. Proteins were purified using HisTrap^™ HP Crude columns (GE Health) according to the manufacturer’s instruction. The buffer was exchanged to 50 mM Tris-HCl buffer (pH 7.4), and glycerol was added to a final concentration of 20% before storage at– 20°C. The enzymes were stable for several weeks under the storage conditions. Finally, protein concentrations were measured using the Bradford assay.