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Volumn 271, Issue 5252, 1996, Pages 1086-1091

The immunological evolution of catalysis

Author keywords

[No Author keywords available]

Indexed keywords

CATALYTIC ANTIBODY;

EID: 0029991227     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5252.1086     Document Type: Article
Times cited : (213)

References (58)
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    • note
    • 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.
  • 19
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    • note
    • 22 distinct nine single-step somatic mutants of 48G7.
  • 21
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    • 6 Cells
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    • note
    • 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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    • C Chothia et al., Nature 342, 877 (1989).
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    • National Institutes of Health, Washington, DC
    • E. Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Washington, DC, 1991), _, T. T. Wu, M. Reid-Miller, H. M Perry, K. S Gottesman, Sequences of Proteins of Immunological Interest (Government Printing Office, Bethesda, MD, 1991).
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    • note
    • H coding region are somatic mutations.
  • 47
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    • note
    • D.
  • 48
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    • note
    • d derived by Scatchard and kinetic analyses.
  • 49
    • 13344285560 scopus 로고    scopus 로고
    • note
    • 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.
  • 55
    • 13344278178 scopus 로고    scopus 로고
    • note
    • 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.
  • 58
    • 13344285559 scopus 로고    scopus 로고
    • note
    • 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).


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.