Two General Methods for the Isolation of Enzyme Activities by Colony Filter Screening

We describe two general methodologies, based on filter-sandwich assays, for isolating enzymic activities from a large repertoire of protein variants expressed in the cytoplasm of E. coli cells. The enzymes are released by the freezing and thawing of bacterial colonies grown on a porous master filter and diffuse to a second "reaction" filter that closely contacts the master filter. Reaction substrates can be immobilized either on the filter or on the enzyme itself (which is then, in turn, captured on the reaction filter). The resulting products are detected with suitable affinity reagents. We used biotin ligase as a model enzyme to assess the performance of the two methodologies. Active enzymes were released by the bacteria, locally biotinylated the immobilized target substrate peptide, and allowed the sensitive and specific detection of individual catalytically active colonies. [on SciFinder (R)]


Published in:
Chemistry & Biology, 9, 3, 383-390
Year:
2002
Keywords:
Note:
CAN 137:244111
9-4
Biochemical Methods
Institute of Pharmaceutical Sciences,Swiss Federal Institute of Technology Zurich,Zurich,Switz.
Journal
1074-5521
written in English.
134712-61-1 (Biotin ligase) Role: ANT (Analyte), ANST (Analytical study) (two general methods involving biotinylation of immobilized peptide substrates permit detection of E. coli enzyme activities by colony filter screening)
Laboratories:




 Record created 2008-11-17, last modified 2018-09-13


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