Ethanolamine

Surface Plasmon Resonance (SPR) experiments were performed at 25 °C on a Biacore 3000 apparatus (GE Healthcare Life Sciences, Uppsala, Sweden) on CM5 sensorchips (GE Healthcare). Capture mAbs were immobilized at 10 μg/mL by amine coupling using a mixture of N-hydroxysuccinimide and N-ethyl-N′-dimethylaminopropyl carbodiimide, according to the manufacturer’s instructions (GE Healthcare), after a 20-fold dilution in sodium acetate buffer (10 mM, pH 5). Then, ethanolamine (1 M, pH 8.5, GE Healthcare) was injected to deactivate the sensor chip surface. Purified HLA-A*02:01 containing the Pp65₄₉₅ peptide were injected at various dilutions over the capture antibodies 180 s at 40 μL/min. A flow cell left blank was used for referencing of the sensorgrams.

To verify the affinity and kinetics between sitagliptin and the novel target, we performed this experiment using Biacore T200 (GE Healthcare) instrument based on SPR, and used GraphPad Prism 8.0 software to visualize the data results (18 (link), 19 (link)).
Firstly, the amino acid sequence of the target protein was searched by Uniprot database, and the sequence was imported into Expasy (https://web.expasy.org/) website to calculate the isoelectric point of the protein (20 (link)). Then, the preconcentration experiment was carried out to determine the optimal coupling conditions. The target protein was coupled to CM5 chip (GE Healthcare) using the Immobilization module in Biacore T200 Control Software, and the reference channel was set to deduct the background. The chip was activated by EDC/NHS (GE Healthcare), and the uncoupling site was blocked by ethanolamine (GE Healthcare).
sitagliptin (HPLC ≥ 98%, Shanghai yuanye Bio-Technology Co., Ltd) was dissolved in DMSO (VWR) solution and diluted with PBS-P+ (GE Healthcare) solution to the desired compound concentration (1×PBS-P+, 5% DMSO). The affinity and kinetics of sitagliptin with the novel target protein were tested by LMW kinetics module in Biacore T200 Control Software, and extra wash after injection with 50% DMSO was added.

Pichia pastoris strain KM71H (aox::ARG4, arg4), expression vector pPICZ-A and Zero Blunt TOPO PCR cloning kit were purchased from Invitrogen. A plasmid containing the gene for full-length human STRA6 transcript variant 2 (NM_022369.3) in the pCMV6-XL4 cloning vector, was purchased from OriGene. The pEGFP-N1 plasmid containing eGFP was purchased from Clontech. Zeocin was from Invivogen. DyLight 594 amine-reactive dye was purchased from Pierce. N-dodecyl β-D maltoside (DDM), N-decyl β-D maltoside (DM) and Fos-Choline-12 (FC-12) were from Anatrace. Cholesteryl hemisuccinate, N, N-dimethyldodecylamine N-oxide (LDAO) and dodecyl nonaoxyethylene ether (C12E9) were from Sigma. HRV 3C protease was from Sino Biological. Polyclonal antibody raised against full-length human STRA6 (#H00064220-D01P) was from Abnova. The Biacore 3000 and instrument-specific consumables (CM5 sensor chip, ethyl(dimethylaminopropyl)-carbodiimide, N-hydroxysulphosuccinimide and ethanolamine) were obtained from GE Healthcare.

Experimental data sets were generated with a BIACORE T100 optical biosensor equipped with research-grade CM5 sensor chips (GE Healthcare, Baie d’Urfe, QC). HBS-EP buffer, acetate buffer and ethanolamine were purchased from GE Healthcare. N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), carbonic anhydrase isozyme II (CAII) that had been purified from bovine erythrocytes, 4-carboxybenzenesulfonamide (CBS), sulfanilamide, 1,3-benzenedisulfonamide (BDS), Sulpiride, Furosemide, 5-dimethyl-amino-1-naphthalene-sulfonamide (DNSA), dimethyl sulfoxide (DMSO) and phosphate buffer saline (PBS, 10 mM, pH 7.4) were purchased from Sigma-Aldrich Canada Ltd (Oakville, ON).

All experiments were performed on a Biacore T200 (GE healthcare).
To capture the streptavidin on the Biacore AU chip (GE healthcare, UK), a thoroughly cleaned Au chip was first functionalized with 11-mercaptoundecanoic acid (0.1 M in ethanol). The surface was then activated with a mixture of 0.2 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 0.05 M N-hydroxysuccinimide (NHS) at a flow rate of 10 µL min⁻¹ for 7 min. Streptavidin (0.1 mg mL⁻¹ in PBS) was subsequently flowed across the surface to an immobilization level of 2000 RU. Ethanolamine (GE Healthcare; 1M, pH = 8.5; 10 µL min⁻¹ for 7 min) was used to block any unreacted activated esters. CXCR4 ACMs (0.01 mg mL⁻¹ in running buffer) were then immobilized at 5 µL min⁻¹ to the desired immobilizations level. BSA (5 mg mL⁻¹) was added to the running buffer to reduce nonspecific binding.
CXCR4 VLPs (Integral Molecular, PA) were captured by direct amine-coupling to the carboxylic acid-functional surface. The VLPs (1∶100 dilution of 400 units in PBS) were then flowed across the surface, resulting in the immobilization of the VLPs. Immobilization level was 5000 RU. Active ester groups were blocked with Ethanolamine (1 M, pH = 8.5).