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Expression of the resulting chimeric Fab yields approximately 2 mg of Fab per liter when cells are grown and induced with limiting phosphate in shake flasks and approximately 30 to 300 mg of Fab per liter when cells are grown in a 2-liter fermentor with limiting phosphate. Electrospray mass spectrometry confirmed that the antibody was fully processed and disulfide-linked (predicted 48G7 mass: 47,219.93 kD, measured 48G7 mass: 47,218.23 ± 1.6 kD). Reactions for the catalytic assays were initiated by the addition of 10 μl of a freshly prepared stock solution of substrate 1 in acetonitrile to 490 μl of antibody solution at 0.2 to 1 0 mg/ml in kinetics buffer (KB consists of 20 mM tris, pH 8.2, and 50 mM NaCl) at 27°C. Hydrolysis rates were measured by absorbance at 400 nm.
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13344276788
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tree = 28.7 percent). All the CDR loops were deleted, simulated-annealed omit maps were computed, and the loops were rebuilt into the electron density. Topology and parameter files were constructed for the hapten, which was refined with the program X-PLOR followed by Powell minimization, simulated annealing, and model building.
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46
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13344271111
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note
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H coding region are somatic mutations.
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47
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13344270566
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note
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D.
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48
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13344278179
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note
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d derived by Scatchard and kinetic analyses.
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49
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13344285560
-
-
note
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H (48G7) Fab fragment was concentrated to 15 mg/ml in a buffer consisting of 100 mM NaCl, 10 mM tris, pH 8 0,. 1.0 mM methionine, 0.5 mM EDTA, 0.3 mM sodium azide; hapten, when present, was at a concentration of 1 mM. Crystallization screens for the Fab fragment (53) were set up in hanging drops at 4°C, with each drop consisting of 2 μl of protein or protein and hapten mixed with 2 μ1 of mother liquor, and each well containing 1 ml of mother liquor. Crystals grew within a few days, and continued to appear for several weeks. Several different crystal morphologies were seen, but invariably the only crystal type that diffracted adequately for data collection were platelike crystals having ill-defined faces and longaxis dimensions in excess of 0.5 mm. The following optimal crystallization conditions were determined for 48G7 Fab (15 mg/ml) with hapten (1 mM): 100 mM sodium acetate, 100 mM sodium cacodylate, pH 7.0, 27 percent PEG 3300.
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55
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13344278178
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note
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H inserts were digested with Mlu I and Kpn I and Mlu I and Apa I, respectively, and cloned into pDEl440, a derivative of pMY61 containing an M13 origin of replication.
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13344285559
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note
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We thank M Davis for use of the BlAcore; D. Henner, S. Bass, and B Snedecore (Genentech) for the plasmids pMY-55, -60, and -61; D. King for performing electrospray mass spectrophotometry; Stanford Synchrotron Radiation Laboratory for data collection time and H. Morimoto (National Tritium Labeling Facility) for assistance synthesizing tritiated hapten 3 Supported in part by the National Institutes of Health (RO1-Al24695), the Howard Hughes Medical Institute, a Damon Runyon-Walter Winchell Cancer Research Fund Fellowship (P A.P.), a Howard Hughes Predoctoral Fellowship (P.L Y.), and the Irvington Institute for Medical Research (J.J.B.) The coordinates have been deposited in the Brookhaven Protein Data Bank (PDB, 11GAF).
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