Fluorescent assay for polymerization of purified bacterial FtsZ cell-division protein.
Septum formation in Escherichia coli is a complex cascade of interactions among cell-division proteins. The tubulin-like FtsZ division protein localizes to the division site and serves a cytoskeletal role during septum formation. A novel fluorescent-based 96-well format filter assay has been developed to measure the polymerization of FtsZ. A mixture of monomers and aggregates (38 to approximately 200 KDa in range) of purified wild-type FtsZ and a fluorescently tagged derivative of FtsZ protein in stoichiometric ratio passes through a 0.2-microm filter membrane, while polymerized FtsZ is retained on the filter. Addition of the SulA protein to the assay leads to rapid disassembly of existing FtsZ polymers, demonstrating its natural regulatory effect on FtsZ under the assay conditions. This assay is sensitive (requiring 2 microM FtsZ or less) and facilitates high-throughput screening of factors affecting FtsZ polymerization.
Department of Endocrinology and Chemical Biology, Building 50G-246, and Biologics Research, 80Y-325, Merck Research Laboratories, Rahway, NJ 07065-0900, USA.
Dorina
D
Trusca
David
D
Bramhill
D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001704:Biopolymers / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D002122:Calcium Chloride / Q000494:pharmacology; D002455:Cell Division; D003598:Cytoskeletal Proteins; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D019793:Fluorescein / Q000737:chemistry*; D005453:Fluorescence; D020558:GTP Phosphohydrolases / Q000378:metabolism; D009154:Mutation; D011189:Potassium Chloride / Q000494:pharmacology; D020836:Protein Structure, Quaternary / Q000187:drug effects; D017186:Titrimetry
D001426:Bacterial Proteins; D001704:Biopolymers; D003598:Cytoskeletal Proteins; D029968:Escherichia coli Proteins; C071525:FtsZ protein, Bacteria; C053139:sulA protein, E coli; D011189:Potassium Chloride; D020558:GTP Phosphohydrolases; D002122:Calcium Chloride; D019793:Fluorescein