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Renal Drug-Metabolizing Enzymes in Experimental Animals and Humans
Published in Robin S. Goldstein, Mechanisms of Injury in Renal Disease and Toxicity, 2020
Much of the early work relied on differences in substrate specificity to determine different isoenzymes. Today, certain model substrates are still used as markers of certain subunits, along with HPLC or fast protein liquid chromatography (FPLC) separation techniques and specific antibodies. l-Chloro-2-4-dinitrobenzene is a general substrate with good activity with all subunits except 5-5 (Table 3). Other substrates show marked specificity, but it is not absolute; for example, 1,2-dichloro-3-nitrobenzene is more active with 3-3 and 6-6 subunits, 1,2-epoxy-3 (p-nitrophenoxy) propane with the 5-5 subunit, 2-hydroxynon-2-enal with the 8-8 subunit, and A5-androstene-3,17-dione with the 1-1 subunit (Table 3).
Alphafetoprotein Down-Regulates ia Antigen Expression on Thyroid Cells
Published in Gérard Chaouat, The Immunology of the Fetus, 2020
Pierre Bobé, Jean Salamero, Nicole Kiger, Jeannine Charreire
Mouse AFP was isolated from 18-d-old C3H(H-2)k mouse fetuses homogenized in PBS, according to the technique of Uriel et al.34 The homogenate was centrifuged (17,000 g, 20 min, 4°C) and the supernatant was dialyzed extensively against sodium acetate:acetic acid buffer (0.05 M, pH 5.4), prior to a CM Sephadex® C-50 (Pharmacia, Fine Chemicals, Uppsala, Sweden) chromatography. Afterwards, the supernatant was concentrated and dialyzed against a Tris-HCl buffer (0.01 M, pH 8) in a CH2 Amicon apparatus (Amicon, Paris). The fraction was then subjected to two different purification procedures. DEAE Trisacryl M (IBF, Villeneuve la Garenne, France) chromatography in Tris-HCl buffer, pH 8, with a molarity gradient ranging from 0.1 to 0.3 M. Each peak eluted was tested by Ouchterlony plating using specific rabbit anti-mouse AFP or anti-mouse serum albumin (MSA) (kindly provided by Dr. Uriel, I.R.S.C, Villejuif, France). AFP was eluted at a molarity of 0.2 M (Peaks 10 and 11) with a yield of approximately 15%, just before fetal mouse albumin (FMA) (Peaks 12 and 13).Fast protein liquid chromatography (FPLC, Pharmacia) using a Mono Q HR 5/5 column (anion exchanger) eluted in Tris-HCl buffer (0.1 M, pH 7) with a 2-M NaCl gradient. AFP was eluted at a 0.6 to 0.7 M NaCl molarity (Peaks I and II) with a yield of approximately 15%, just before the FMA (Peak IIT).
General and Practical Aspects of Membrane Protein Crystallization
Published in Hartmut Michel, Crystallization of Membrane Proteins, 1991
The separation methods for membrane proteins are essentially the same as for soluble proteins. It is normally sufficient to work at detergent concentrations slightly above the CMC. Frequently ion-exchange chromatography which is based on surface charges of the protein is the method of choice, especially in the modem FPLC-version. Both anion and cation exchangers can be used but neither can be used with ionic detergents. Zwitterionic detergents also possess disadvantages due to electrostatic interactions as a consequence of the strong dipole moment at their polar head groups. The column must be equilibrated with the detergent containing buffer prior to its use. The Pharmacia corporation (Uppsala, Sweden) provides a complete reference list upon request. Extremely powerful methods are isoelectric focusing, where the commercially available preparative scale equipment can be used especially in connection with alkylglucosides, alkylthioglucosides, and alkylmaltosides as detergents, and chromatofocusing. In both methods proteins are separated according to their isoelectric points. In the chromatofocusing technique the proteins are bound to special ampholytic column materials and eluted with a focusing pH-gradient. In addition, isoelectric focusing is the best analytical method to test purity and homogeneity of the final membrane protein preparation. Isopycnic sucrose density gradient centrifugation is also very useful for the purification of membrane proteins, in contrast to the purification of soluble proteins. The density of the pure proteins is more or less the same. Since we purify mixed proteindetergent micelles, isopycnic ultracentrifugation separates membrane proteins according to their detergent to protein ratio. Pigments, when present, also influence the density. Solubilized chlorophyll proteins can be easily purified by this method. Its power, however, has not been fully explored with other membrane proteins.
Enhanced anti-cancer effect using MMP-responsive L-asparaginase fused with cell-penetrating 30Kc19 protein
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2022
Jina Ryu, Sung Jae Yang, Boram Son, Haein Lee, Jongmin Lee, Jinmyoung Joo, Hee Ho Park, Tai Hyun Park
The constructed vectors were used to transform E. coli BL21 (Novagen, Madison, WI, USA), and cells were grown in LB-ampicillin medium at 37 °C in 200 rpm shaking incubator. After induction using isopropyl 1-thio-β-d-galactopyranoside (1 mM) with optical density of 600, cells were further incubated at 37 °C for 4 h. Harvested cells were resuspended in binding buffer (20 mM Tris–HCl, 0.5 M NaCl, 20 mM imidazole, pH 8.0), and disrupted by sonication. After centrifugation (12,000 rpm, 30 min), proteins were loaded to a HisTrap HP column (GE healthcare, Uppsala, Sweden), then non-binding proteins were washed using wash buffer (20 mM Tris–HCl, 0.5 M NaCl, 50 mM imidazole, pH 8.0). Finally, target proteins were purified by fast protein liquid chromatography (FPLC, GE healthcare) using elution buffer (20 mM Tris–HCl, 0.5 M NaCl, 350 mM imidazole, pH 8.0). HisTrap Desalting column (GE healthcare) was used for dialysis through 20 mM Tris–HCl buffer (20 mM Tris–HCl, 0.5 M NaCl, pH 8.0). The purified proteins were quantitated using a Micro BCA kit (Thermo Scientific Inc., Rockford, IL, USA) with a standard bovine serum albumin solution, and then stored at −70 °C for further use.
A strategy for the efficient construction of anti-PD1-based bispecific antibodies with desired IgG-like properties
Published in mAbs, 2022
Jie Zhao, Liangfeng Jiang, Haodong Yang, Lan Deng, Xiaoqing Meng, Jian Ding, Sixing Yang, Le Zhao, Wei Xu, Xiaolong Wang, Zhenping Zhu, Haomin Huang
FreeStyle™ 293-F cells (Thermo Fisher Scientific, Cat#R79007) were cultured in serum-free medium. Transient transfection was performed by co-transfection of expression vectors encoding a heavy chain or a light chain individually into FreeStyle™ HEK293-F cells using 1 μg/ml 25 KDa linear polyethylenimine (Polysciences, Inc.). One day after transfection, valproic acid (Sigma) was added to cell culture at a final concentration of 3 mM. On day 2 post-transfection, medium comprising 10% GlutaMAX, 10% 400 g/L glucose and 80% freestyle 293 medium was added to the cell culture at 10% of the total volume. Conditioned medium was collected 5–6 days after transient transfection. Antibodies in the culture media were purified by MabSelect SuRe affinity columns (GE Healthcare) on an AKTA Avant 25 fast protein liquid chromatography (FPLC) System. The columns were equilibrated with buffer A (25 mM sodium phosphate, 150 mM sodium chloride, pH = 7.0) prior to use. The culture media containing antibodies were then applied to the columns followed by elution with Buffer B (100 mM sodium citrate, pH 3.5) to collect the desired proteins. Collected proteins were neutralized with 1 M Tris (pH 9.0), which were then dialyzed against phosphate-buffered saline (PBS). Finally, the purity of the samples was analyzed on a SEC-high performance liquid chromatography (SEC-HPLC).
Antibody-mediated delivery of LIGHT to the tumor boosts natural killer cells and delays tumor progression
Published in mAbs, 2021
Marco Stringhini, Jacqueline Mock, Vanessa Fontana, Patrizia Murer, Dario Neri
The DNA sequence encoding murine LIGHT extracellular domain (amino acids 87–239) in a single-chain format (in which three LIGHT subunits were genetically linked together by a Glycine codon) including an N-terminal (SSSSG)3-linker, was purchased from Eurofins genomics. The LIGHT gene was fused by PCR assembly to the C-terminal end of various formats of the F8 antibody via its 15 amino acids linker. The resulting genes were cloned into the mammalian vectors pcDNA3.1+ (for F8 in scFv-Fc and diabody formats) or pMM137 (for F8 in IgG format) by restriction enzymes digestion and ligation, followed by amplification in TG1 electrocompetent E. coli bacteria. pMM137 was kindly provided by Philochem AG and has been described elsewhere.47 Fusion proteins were produced in CHO-S by transient gene expression as already described.48,49 Both “low density” (LD)48 and “high density” (HD)49 protocols were used. Proteins were purified to homogeneity by protein A affinity chromatography and characterized by size exclusion chromatography on a Äkta Pure FPLC system (GE Healthcare) with a Superdex S200 10/300 increase column (GE Healthcare) and by SDS-PAGE.