Abstract
Immunoassays of dnaA protein in extracts from five strains showed a rather constant abundance relative to cell mass, with a variation of 800-2100 molecules/cell; overproducing cells contained 100-fold that number. About half of the dnaA protein in wild type cells was solubilized by a lysis procedure. Within the insoluble fractions, dnaA protein was identified by its characteristic high-affinity binding of ATP. An improved, rapid procedure for purifying dnaA protein from overproducing cells appears to depend on its coprecipitation with phospholipids and depends on solubilization by guanidine HCl. The procedure, with a 5-fold increased yield, also eliminates a potent ATPase contaminant. Purified dnaA protein, unlike dnaB and dnaC proteins, binds to phospholipid vesicles as judged by analysis on sucrose gradient centrifugation.
Highlights
Immunoassaysof dnaAprotein in extracts from five strains showed a rather constant abundance relative to cell mass, with a variation of 800-2100 molecules/ cell; overproducingcells contained 100-fold that number
About half of the dnaA protein in wild type cells was solubilized by a lysis procedure
DnaA protein plays a crucial role in the initiation of Escherichia coli chromosomal replication
Summary
Reagents-Sources were: ATP, deoxyribonucleosidetriphosphates, and HEPES,' Pharmacia P-L Biochemicals;lysozyme(egg white), GTP, CTP, UTP, and Tricine, Sigma; [w3'P]ATP (410 Ci/mmol), [L~-~'P]TT(8P00 Ci/mmol), and [32P]orthophosphoricacid (carrier free), Amersham Corp.; [y-32P]ATP(6000 Ci/mmol), Du Pont-New England Nuclear; '"I-protein A (33.4 mCi/mg), ICN Radiochemicals; Bio-Rex 70, Bio-Rad. Purified dnaA protein (0.23 mg)was diluted 10-fold with a buffer (50 mM Tricine.KOH, pH 8.25 at 1M, 0.5 mM m a ~ e s i u macetate, 0.3 mM EDTA, 5 mM DTT, 10 mM (NH,),SO,, 17% (v/v) glycerol, 0.005% Triton X-100) and filtered through a nitrocellulose membrane (Millipore HA 0.45 PM, 3.5-cm diameter). Anti-dnaA protein antibody was recovered by washing the filter by suction with 5 ml of 0.2 M glycine.HCl, pH 2.8, neutralized with NaOH, adjusted to a 10%concentration of fetal calf serum and stored a t -80 "C. The final membrane fractions (4 pg of protein, 0.24 pmol of dnaA protein, determined by immunoblot analysis) were suspended in 30 pl of 50 mM Tricine.KOH, pH 8.25 a t 1 M, 12.5 mM magnesium acetate, 0.3 mM EDTA, 20% (v/v) glycerol, 0.007% Triton X-100, 7.
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