title: Purification of F plasmid-encoded native TraC from Escherichia coli by affinity chromatography on calmodulin Sepharose.
abstract: We have enriched several native bacterial proteins from Escherichia coli by chromatography on the immobilized eukaryotic Ca(2+)-binding protein, calmodulin. These bacterial proteins bound in a Ca(2+)-dependent manner to calmodulin, and were released by the addition of the Ca(2+)-chelator, EGTA, similar to many eukaryotic calmodulin-binding proteins. One of the bacterial proteins, F factor-encoded TraC, was purified to apparent homogeneity by an additional chromatographic step, anion exchange chromatography on MonoQ. Experiments with four chemically distinct calmodulin antagonists (R24571, Compound 48/80, melittin, and W7) showed that all of these substances inhibited the binding of purified TraC to calmodulin at effective concentrations comparable to those required for inhibiting in vitro binding of eukaryotic calmodulin-binding proteins. Three further bacterial proteins were identified as calmodulin-binding proteins: SecA, GlpD, and GlpC. We suggest that also these native bacterial proteins might be isolated by the unusual purification procedure including affinity chromatography on calmodulin Sepharose. Whether the identified proteins bind to, and are regulated by, putative bacterial calmodulin-like proteins in Escherichia coli remains to be established.
authors.affiliation: Fakultät für Biologie, Universität Konstanz, 78457 Konstanz, Germany. Electronic address: Simon.Hellstern@hs-furtwangen.de.
authors.forename: Simon
authors.initials: S
authors.lastname: Hellstern
authors.affiliation: Fakultät für Biologie, Universität Konstanz, 78457 Konstanz, Germany.
authors.forename: Rupert
authors.initials: R
authors.lastname: Mutzel
mesh_terms: D000595:Amino Acid Sequence; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D036781:Fimbriae Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives
chemical_list: D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D029968:Escherichia coli Proteins; C074790:traC protein, Plasmid F; D036781:Fimbriae Proteins; D012685:Sepharose
keywords: Affinity chromatography; Binding protein; Calmodulin; Calmodulin Sepharose; Calmodulin antagonist; TraC