Cm sepharose fast flow

For purification, an approximately 15 g aliquot of cell pellet were resuspended in 50 mM potassium phosphate pH 7.8, adjusted by altering the ratio of dibasic (K₂HPO₄) and monobasic forms (KH₂PO₄) to 91 and 9%, respectively (Cold Spring Harbor Laboratory, 2006 ▸ ). Lysis was conducted with an Emulsiflex-C3 and the clarified lysate was incubated at 60°C for 1 h to precipitate contaminant proteins, which were removed by centrifugation. Soluble protein was dialyzed against 5 mM potassium phosphate pH 7.8 and applied to pre-swollen diethylaminoethyl (DE52) cellulose resin (GE Healthcare). The protein–resin slurry was rocked for 1 h at 10°C and poured into a Büchner funnel with Whatman No. 4 filter paper while under vacuum. Once dry, the resin was washed with 5 mM potassium phosphate pH 7.8 and the protein was then eluted with 100 mM potassium phosphate pH 7.8. The eluted protein was dialyzed against 2.5 mM 2-(N-morpholino)ethanesulfonic acid (MES) pH 5.5, applied onto a carboxymethyl (CM) Sepharose Fast Flow (GE Healthcare) column, eluted with a sodium chloride gradient and concentrated to 21 mg ml⁻¹ using 5 kDa molecular-weight cutoff concentrators. The concentration was measured using a NanoDrop ND-1000 spectrophotometer using an extinction coefficient of 40 500 M⁻¹ cm⁻¹ at 280 nm (Lévêque et al., 2001 ▸ ). The purifications of hydrogenated and deuterated protein were identical.

Porcine [Gly²] GLP-2 (HGDGSFSDEMNTVLDNLATRDFINWLLHTKITDSL, > 98%) was synthesised by Chinese Peptide Company (Hangzhou, China). Monomethoxy PEG–succinimidyl propionate (mPEG–SPA; molecular weight (MW) = 5 kDa) was purchased from Beijing Kaizheng Biotech Development Co., Ltd. (Beijing, China). The ion-exchange chromatography (IEC) resin and column used were CM Sepharose Fast Flow (GE Healthcare Bio-Science AB, Uppsala, Sweden). Ultrafiltration membranes with a MW cutoff of 3000 were purchased from Millipore Corporation (Billerica, MA, USA). Dextran sodium sulphate (DSS) (MW = 36 000–50 000) was purchased from MP Biomedicals China (Shanghai, China). RNAiso Plus, PrimeScript®RT-reagent with gDNA Eraser Kit and SYBR® Premix Ex TaqTM were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). RNA locker was purchased from TIANDZ (Beijing, China). Unless otherwise specified, all other chemicals and reagents were of analytical grade from Sigma–Aldrich and Fluka (Milan, Italy).

FNR-675 N-hydroxysuccinimide (NHS) ester was purchased from BioActs (Incheon, Korea). Superdex^TM 200 (10/300); CM sepharose^® Fast Flow and DEAE sepharose^® Fast Flow were bought from GE Healthcare (Piscataway, NJ, USA). The 2′,7′-dichlorofluorescein diacetate (DCF-DA) and MitoSOX Red were obtained from Molecular Probes Inc., (Eugene, OR, USA). Histatin 5 and melittin were synthesized by using a microwave peptide synthesizer (Discover Bio^TM, CEM Co., Matthews, NC, USA) via solid phase method. All other chemicals were of reagent grade and all solvents were HPLC grade.