Development of a High-Throughput Screen for Protein Catalysts: Application to the Directed Evolution of Antibody Aldolases

Angewandte Chemie - International Edition

Volumn 42, Issue 48, 2003, Pages 5971-5973

  Gildersleeve, Jeff ^a   Varvak, Alex ^a   Atwell, Shane ^a   Evans, Doug ^b   Schultz, Peter G ^a  

^a SCRIPPS RESEARCH INSTITUTE   (United States)

^b GENOMICS INSTITUTE OF THE NOVARTIS RESEARCH FOUNDATION   (United States)

Author keywords

Aldol reaction; Antibodies; Directed evolution; High throughput screening; Proteins

Indexed keywords

CATALYSIS; ENZYMES;

PROTEIN EXPRESSION LEVELS; PROTEIN SELECTIVITY;

ANTIBODIES;

ALDOLASE ANTIBODY; ENZYME ANTIBODY; UNCLASSIFIED DRUG;

ARTICLE; CATALYSIS; ENZYME ACTIVITY; ENZYME ASSAY; HIGH THROUGHPUT SCREENING; LABORATORY AUTOMATION; PROTEIN ANALYSIS; PROTEIN DEGRADATION; PROTEIN EXPRESSION; PROTEIN PURIFICATION;

ALDEHYDE-LYASES; ANTIBODIES, CATALYTIC; CATALYSIS; COMPLEMENTARITY DETERMINING REGIONS; DIRECTED MOLECULAR EVOLUTION; ELECTROPHORESIS, POLYACRYLAMIDE GEL; ENZYME-LINKED IMMUNOSORBENT ASSAY; PROTEINS;

EID: 0346724593     PISSN: 14337851     EISSN: None     Source Type: Journal    
DOI: 10.1002/anie.200352117     Document Type: Article

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12. Detailed procedures for a typical high-throughput screen, the construction of the expression vector, the construction of the antibody libraries, and characterization of the antibodies can be found in the Supporting Information.
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18. The 26 libraries were constructed in four groups, based on the location of the randomized residue: light CDR1, light CDR3, heavy CDR1, and heavy CDR3 (see Supporting Information). The four groups were transformed separately and screened as described in the supporting information with a unique clone in each well. Plasmid DNA from each hit identified in the screen was obtained by isolating the DNA from the corresponding starter culture well.