Mechanisms of Antibiotic Resistance in Acinetobacter spp. — Genetics of Resistance
E. Bergogne-Bénézin, M.L. Joly-Guillou, K.J. Towner in Acinetobacter, 2020
The above technique readily demonstrates the difference between the four chromosomal ß-lactamases, but is time consuming and very expensive. The results that it gives can be confirmed by separation of the ß-lactamases with ion-exchange chromatography. The basis of separation is by overall charge and is thus similar to IEF. Fast-Protein Liquid Chromatography (FPLC) allows rapid separation with the cation exchange column MONO S and the anion exchange column MONO Q. The fractions are tested for ß-lactamase activity with nitrocephin. FPLC separation showed clear differences between C-lactamases ACE-1, ACE- 3 and ACE-4, and suggested that there may be some relation between ACE-1 and ACE-2. Final confirmation of the distinct nature of these enzymes came from separation of the FPLC/ion exchange ß-lactamase peaks on a PAGE minigel. This gel system was devised to resolve basic proteins on polyacrylamide by Olsson and Tooke (1988), and was adapted to include a Phastgel Homogeneous 20 (SDS-free), run with reverse polarity, with visualisation of the E-lactamases by nitrocephin (Hood and Amyes, 1991).
Exchange Factors
Juan Carlos Lacal, Frank McCormick in The ras Superfamily of GTPases, 2017
In the laboratory of Tohru Kamata an exchange factor has been found in detergent extracts of bovine brain membranes.15,16 This was initially identified when it was found that Triton® X-100 extracts of membrane preparations from bovine brain, but not cytosolic extracts, were capable of increasing the rate of loss of [3H]GDP from purified wild-type p21ras in the presence of unlabeled nucleotide. Such activity was also found in similar preparations from a wide range of tissues and also from NIH 3T3 fibroblasts and Xenopus laevis oocytes. Upon purification, the exchange activity ran on a Superóse 12 FPLC column with an apparent relative molecular weight of 100,000. Further purification to homogeneity revealed that the activity was associated with a protein that ran as a single band on an SDS-polyacrylamide gel with a molecular weight of 35,000. This protein was termed “rGEF” for ras guanine nucleotide exchange factor.
Renal Drug-Metabolizing Enzymes in Experimental Animals and Humans
Robin S. Goldstein in 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).
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.
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.
Early events in light chain aggregation at physiological pH reveal new insights on assembly, stability, and aggregate dissociation
Published in Amyloid, 2021
Pinaki Misra, Marina Ramirez-Alvarado
Proteins were expressed in Escherichia coli BL21 (DE3) gold competent cells (Agilent Technologies, Santa Clara, CA) as reported previously [12,13,19]. AL-09 was extracted from the insoluble fraction (using 6 M urea) while all other proteins (κIO18/O8, κIO18/O8 Y87H, AL-09 H87Y, AL-12, and AL-12 R65S) were extracted from the periplasmic space by breaking the cells through one freeze thaw cycle using PBS buffer at pH 7.4 as described previously [13,16,17]. Purification of all proteins was conducted on an AKTA FPLC (GE Healthcare, UK) system with a HiLoad 16/60 Superdex 75 size exclusion chromatography (SEC) column using 10 mM Tris-HCl at pH 7.4 as mobile phase. Protein content verification and protein size verification, were done by UV-Spectrophotometry (Absorbance ratio at 260/280 nm of less than 1) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) respectively. Secondary and tertiary structure and qualitative assessment of the purified fractions were done using far UV-CD spectra and thermal unfolding/refolding experiments at 217 nm on a CD spectropolarimeter (JASCO J 810, JASCO Corporation, Japan), as reported previously [10,11,20]. Protein concentration was determined by UV absorbance at 280 nm using extinction coefficient calculated from amino acid sequence as follows: ε = 14890 M−1·cm−1 for κI O18/O8 and AL-09 H87Y, and ε = 13610 M−1·cm−1 for κIO18/O8 Y87H, AL-09, AL-12, and AL-12 R65S.
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