Hitrap sp sepharose fast flow column

The small-scale refolding screen analyses were performed with a final volume of 1–5 mL. For rapid dilution, the purified monomeric MB109 was diluted directly into cold refolding solutions, mixed rapidly by vortexing and incubated at 4°C. To visualize the progress of refolding, visible protein aggregates in the sample solutions were spun down first and the supernatants were diluted in an acidic buffer solution to stop disulfide reshuffling or redox reaction and concentrated to run on non-reduced 12% SDS-PAGE. The densitometry of the functional dimer bands on the SDS-PAGE gel images was analyzed by using the GeneTools image analysis software (Synoptics, UK). To purify the refolded MB109 functional dimer, the pH of the refolded sample was titrated directly to 3.2 and subjected to gel filtration chromatography by HiLoad 16/60 Superdex™ 75 and 200 columns (Figure 4B). For cation exchange chromatography, 1-mL HiTrap SP Sepharose Fast Flow column (GE Healthcare) was used with the elution salt gradient ranging from 0 to 1 M NaCl (Figure 4C).

Reaction mixtures with WT enzyme or E87Q were incubated for 1 day with 2 mm streptomycin and 10 mm ATP in a buffer containing 50 mm HEPES, pH 7.5, 160 mm NaCl, 10 mm MgCl₂, 1 mm DTT, and pyrophosphatase at room temperature with gentle agitation. Fresh AadA and pyrophosphatase were added at four time points during the incubation to end up with a final concentration of 0.03 mg/ml and 1.5 units/ml, respectively. After incubation, the enzymes were removed from the reaction mixtures by filtration with a Vivaspin 20 concentrator (polyethersulfone membrane, 10,000-Da molecular weight cutoff, Sartorius), and the antibiotic was washed out from the enzyme by successive dilutions in buffer A (20 mm MES, pH 6.0). 5 ml of filtrate was applied to a 1-ml HiTrap SP Sepharose Fast Flow column (GE Healthcare). Nonreacted ATP and AMP eluted upon wash with buffer A. Adenylated streptomycin was eluted with a 0–25% gradient of buffer B (20 mm MES, pH 6.0, 1 m NaCl) in 20 column volumes, and absorbance was monitored at 260 nm.

The reaction mixture consisted of 50 mM HEPES (pH 8.0) containing 2 mM streptomycin, 10 mM ATP, 160 mM NaCl, 10 mM MgCl₂, and 1 mM DTT. Freshly purified protein and pyrophosphatase were added to the reaction mixture at four 6 h intervals during the 24 h incubation at room temperature with controlled agitation. The final concentration of the protein and pyrophosphatase at the end of 24 h was 0.03 mg/mL and 1.5 units, respectively. The reaction mixture containing protein was removed using a 10 Kilo Dalton (kDa) cutoff concentrator (Merck). The 5 mL of filtrate containing adenylated streptomycin was applied to a 1 mL HiTrap SP Sepharose Fast Flow column (GE Healthcare), and the unbound antibiotics, non-reacted ATPs, and adenosine monophosphates (AMPs) were removed by washing with MES buffer (20 mM MES pH 6.0). The strongly bound adenylated streptomycin was eluted using 20 column volumes of elution buffer (20 mM MES pH 6.0, 1 M NaCl) in a linear gradient manner, and the absorbance was monitored at 260 nm [21 (link)].