China
Africa
Explore chapters and articles related to this topic
Protein-Detergent Micellar Solutions for the Crystallization of Membrane Proteins: Some General Approaches and Experiences with the Crystallization of Pigment-Protein Complexes from Purple Bacteria
Published in Hartmut Michel, Crystallization of Membrane Proteins, 1991
We use gel filtration on Superose 6B to test our final preparations for homogeneity of size (oligomerization). With more than 50 μM Ca++ in the elution buffer, the Ca++-ATPase tends to elute as a mixture of monomers and dimers, while in the absence of Ca++ it is monomelic.
Bispecific antibody CAP256.J3LS targets V2-apex and CD4-binding sites with high breadth and potency
Published in mAbs, 2023
Baoshan Zhang, Jason Gorman, Young D. Kwon, Amarendra Pegu, Cara W. Chao, Tracy Liu, Mangaiarkarasi Asokan, Michael F. Bender, Tatsiana Bylund, Leland Damron, Deepika Gollapudi, Paula Lei, Yile Li, Cuiping Liu, Mark K. Louder, Krisha McKee, Adam S. Olia, Reda Rawi, Arne Schön, Shuishu Wang, Eun Sung Yang, Yongping Yang, Kevin Carlton, Nicole A. Doria-Rose, Lawrence Shapiro, Michael S. Seaman, John R. Mascola, Peter D. Kwong
Light chain expression constructs of CAP256V2LS and PGDM1400 antibody variants were synthesized (Gene Universal Inc.) and cloned into pVRC8400 expression vector. For the antibody production, 0.15 mL of Turbo293 transfection reagent (Speed BioSystems) were mixed into 2.5 mL Opti-MEM medium (Life Technology) and incubated for 5 min at room temperature (RT). 50 μg of plasmid DNAs (25 μg heavy chain and 25 μg of light chain) were mixed into 2.5 mL of Opti-MEM medium in another tube. The diluted transfection reagent was added into Opti-MEM medium containing plasmid DNAs. Transfection reagents and DNA mixtures were incubated for 15 min at RT and added to 40 mL of Expi293 cells (Life Technology) at 2.5 million cells/ml. The transfected cells were cultured in a shaker incubator at 120 rpm, 37°C, 9% CO2 for 5 days. Antibodies in clarified supernatants were purified over 0.5 mL Protein A (GE Health Science) resin in columns. Antibodies were eluted from Protein A columns with a low pH immunoglobulin G (IgG) elution buffer (Pierce) and immediately neutralized with one-tenth volume of 1 M Tris-HCL pH 8.0. The antibodies were buffer exchanged in phosphate-buffered saline (PBS) by dialysis and then the concentration was adjusted to 0.5 mg/ml and filtered (0.22 μm) for neutralization assays. For size-exclusion chromatography analysis, 0.5 mg of 1 mg/ml antibody was injected onto the Superose 6 Increase column (Cytiva Life Sciences) on a BioRad (Hercules, CA) NGC Chromatography System Quest 10. The flow rate was set to 0.5 mL/min, and the mobile phase B was 1 × PBS (Thermo Fisher).
DMSO-tolerant ornithine decarboxylase (ODC) tandem assay optimised for high-throughput screening
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2023
Mingu Gordon Park, Suyeon Yellena Kim, C. Justin Lee
The entire purification procedure was performed at 4 °C. Size-exclusion chromatography of recombinant human ODC homodimer was performed in a ÄKTA go chromatography system (Cytiva, 29383015) equipped with an ultraviolet (UV)-visible detector. Standard (BioRAD, 1511901) or sample solutions were injected into the HPLC system through a Rheodyne injector. The separation was achieved by using Superose® 6 Increase 10/300 GL column (Cytiva, 29091596). The running buffer at a flow rate of 0.5 mL/min was used as a mobile phase to transfer and various proteins mixed in the eluate were discriminated according to their molecular weight. The chromatograms were monitored by UV detection at a wavelength of 280 nm. 0.5 mL of fractions were collected every minute for 1 h. Lastly, the fractions containing pure human ODC homodimers were combined. The purified protein concentration was measured by NanoDrop One spectrophotometer (Thermo Fisher Scientific) using absorbance at 280 nm. The purified recombinant human ODC was stored at −80 °C for long term storage.
Protective antibodies against human parainfluenza virus type 3 infection
Published in mAbs, 2021
Jim Boonyaratanakornkit, Suruchi Singh, Connor Weidle, Justas Rodarte, Ramasamy Bakthavatsalam, Jonathan Perkins, Guillaume B.E. Stewart-Jones, Peter D. Kwong, Andrew T. McGuire, Marie Pancera, Justin J. Taylor
Expression plasmids for His-tagged HPIV3 preF and postF antigens are previously described.25 HPIV3 preF contained the following mutations including two disulfide linkages, Q162C-L168C, I213C-G230C, A463V, and I474V.25 293 F cells were transfected at a density of 106 cells/mL in Freestyle 293 media using 1 mg/mL PEI Max (Polysciences, cat#24765). Transfected cells were cultured for 7 days with gentle shaking at 37°C. Supernatant was collected by centrifuging cultures at 2,500 × g for 30 minutes followed by filtration through a 0.2 µM filter. The clarified supernatant was incubated with Ni Sepharose beads overnight at 4°C, followed by washing with wash buffer containing 50 mM Tris, 300 mM NaCl, and 8 mM imidazole. His-tagged protein was eluted with an elution buffer containing 25 mM Tris, 150 mM NaCl, and 500 mM imidazole. The purified protein was run over a 10/300 Superose 6 size-exclusion column (GE Life Sciences, cat#17–5172–01). Fractions containing the trimeric HPIV3 F proteins were pooled and concentrated by centrifugation in an Amicon ultrafiltration unit (Millipore, cat#UFC805024) with a 50 kDa molecular weight cutoff. Two units of biotinylated thrombin (Millipore, cat#69022) were mixed with each 1 mg of protein overnight to cleave off tags, streptavidin agarose (Millipore, cat#69022) was added for another hour to remove thrombin and the cleaved tags, and the mixture was centrifuged through a PVDF filter (Millipore, cat#UFC40GV25) to remove the streptavidin agarose. The concentrated sample was stored in 50% glycerol at −20°C.