Hiprep 26 10 desalting column

Cells were harvested by centrifugation and suspended in lysis buffer containing 50 mM NaH₂PO₄, 300 mM NaCl, and 10 mM imidazole, pH 8.0. Cells were maintained at 4 °C, disrupted by sonication and centrifuged at 100,000× g for 1 h. The supernatant was added to a 5 mL HisTrap HP column (GE Healthcare, Chicago, IL, USA) and eluted with an imidazole gradient (0–1000 mM). The eluted protein was added to a HiPrep 26/10 Desalting column (GE Healthcare, Piscataway, NJ, USA) in 50 mM Tris HCl, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), pH 8.0 (a buffer favoring Tobacco Etch Virus protease (TEVp) activity). The eluted enzyme was incubated overnight at 30 °C with TEVp at a ratio of 20 RmTIM/1 TEVp (w/w). The digested proteins were then added to a Hi Prep 26/10 Desalting column (GE Healthcare) in a buffer containing 50 mM NaH₂PO₄, 300 mM NaCl, and 10 mM imidazole, pH 8.0, and the eluted sample separated in the HisTrap HP column (GE Healthcare). This last step removes the (His)6-tag-TEVpV from the RmTIM. The enzyme was concentrated to 6 mg/mL with Amicon Ultra filters with 30,000 MWCO (Millipore Co., Bedford, MA, USA), and dialyzed overnight in 100 mM triethanolamine (TEA), 1 mM EDTA, and 1 mM DTT. The purification steps were monitored by SDS-PAGE gels containing 16% acrylamide, and stained with Coomassie blue.

¹H PRE data were gathered at 25 °C on a Bruker AVANCE III-HD 600-MHz spectrometer installed with a cryo-probe. The protein construct Nop15 40-191 K45C no linker was prepared as described above except that the E. coli cells were grown in M9 media containing ¹⁵NH₄Cl. Immediately before paramagnetic labeling with MTSL, TCEP was removed by loading the sample onto a HiPrep 26/10 desalting column (GE) equilibrated with 20 mM Tris HCl, pH 7.5, 100 mM NaCl, and 400 mM arginine/glutamate. The protein was diluted to 40 μM and mixed with 200 μM MTSL for overnight reaction at 4 °C. Unreacted MTSL was removed by loading the sample onto a HiPrep 26/10 desalting column (GE) equilibrated in 20 mM MES, pH 6.0, 400 mM arginine/glutamic acid, and 5% D₂O. The PRE measurements were carried out using a pulse sequence developed by Junji Iwahara (38 (link)). A total of 64 scans were accumulated, and the relaxation time interval was set to 8 ms. Diamagnetic data were collected with the above sample quenched using 2 mM ascorbic acid. The NMR data were processed using NMRPipe (53 (link)) and analyzed using NMRViewJ (54 (link)). The errors were estimated from PRE measurements of two independent samples.

The culture supernatants were collected by centrifugation at 12,000g, 4°C for 10 min and concentrated through Vivaflow 200 membrane of 5-kDa molecular weight cutoff (Vivascience, Germany). The crude enzyme was loaded onto the HiPrepTm 26/10 Desalting column and HiTrapQHP column (GE Healthcare). Fractions containing lipase activity were pooled and concentrated by ultrafiltration (5-kDa molecular weight cutoff) for further characterization. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out with the 5% stacking gel and 12% separation gel. Protein concentration was determined using the Bradford method with bovine serum albumin as the standard.