Arginine Hydrochloride

Recombinant CXCL12 was expressed and purified from E. coli. A pET21-based vector containing residues 22–89 of human CXCL12α (GenBank accession code NM_199168.3) preceded by an enterokinase recognition site and a His₈ tag was transformed in BL21(DE3)pLys cells (Promega). Cells were grown to an optical density of 0.7 in Luria-Bertani (LB) medium, protein expression was induced by the addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 6 h after induction cells were harvested by centrifugation. Cell pellets were suspended in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl) and lysed by sonication. The lysate was centrifuged (6,000 g for 15 min) and the pellet containing CXCL12 inclusion bodies was dissolved in 50 mM Tris, pH 8, 6 M guanidine-HCl, 4 mM DTT (dithiothreitol). The resulting suspension was sonicated and centrifuged (6,000 g for 15 min) and the supernatant was passed over a Ni-NTA agarose resin (Qiagen) using gravity flow. The resin was washed with 50 mM MES, pH 6, 6 M guanidine-HCl, 4 mM DTT and protein was eluted with 50 mM acetate, pH 4, 6 M guanidine-HCl and 4 mM DTT. The eluate was diluted 10-fold with refolding buffer (50 mM Tris, pH 7.5, 500 mM arginine-HCl, 1 mM EDTA, 1 mM glutathione disulfide) and incubated for 60 min with stirring. The refolding mixture was dialyzed in 20 mM Tris, pH 8.0 and 50 mM NaCl. After dialysis, 2 mM CaCl₂ was added and the His₈ tag was cleaved through the addition of enterokinase (New England Biolabs). After cleavage the mixture was added to a Ni-NTA resin and cleaved CXCL12 was eluted with 6 M guanidine and 50 mM MES, pH 6.0. The eluate was bound to a reversed-phase C18 HPLC column (Vydac) (buffer A: 0.1% trifluoracetic acid; buffer B: 0.1% trifluoroacetic acid; 90% acetonitrile) and eluted by linearly increasing the buffer B concentration from 33 to 45%. The resulting pure CXCL12 protein was lyophilized and stored at −80 °C until use.
HRV3C protease was produced in E. coli. A plasmid containing the HRV3C sequence and an N-terminal His tag was transformed into BL21(DE3)pLys cells and plated onto kanamycin-resistant LB agar plates. A single colony was picked and grown overnight in 10 ml LB media supplemented with 30 μg ml⁻¹ kanamycin at 37 °C. The overnight culture was diluted 1:100 in LB/kanamycin and grown to an OD₆₀₀ of 0.6–0.8, at which point protein expression was induced through the addition of 0.5 mM IPTG. At 3 h after inducation, the cells were harvested by centrifugation. Cell pellets were resuspended in 50 mM Tris, pH 8.0, and sonicated for 10 min on ice. The suspension was then centrifuged (50,000 g for 20 min) and pellets were homogenized in lysis buffer (100 mM Na₂H₂PO₄, 10 mM Tris, 6 M urea, pH 8.0). The resulting suspension was centrifuged (50,000 g for 25 min) and the supernatant was incubated for 1 h with Ni-NTA resin. The resin was washed with lysis buffer and protein was eluted with elution buffer (50 mM sodium acetate, 6 M urea, pH 5.0). The eluted sample was then dialyzed twice against dialysis buffer (20 mM MES, 1 mM EDTA, 1 mM DTT, 10% (v v⁻¹) glycerol, pH 6.5), flash frozen and stored at −80 °C until further use.

AST was used to assess first-phase insulin secretion (FPIS) after overnight fasting for at least eight hours. After a baseline blood sample was collected, a 10% (wt/vol.) solution of arginine hydrochloride (5 g) (Shanghai Xinyi Jinzhu Pharmaceutical Co., Ltd., Shanghai, China) was injected intravenously within 30-45 s. Blood samples were obtained at 2, 4, and 6 min after injection (17 (link)). All anti-diabetic therapy was paused during the test.