Over-expression, purification and phosphorylation of the Bacillus subtilis CheA protein

The CheA (Bacillus subtilis) protein was over-expressed in a CheA minus E. Coli strain. Cells induced for the over-expression of CheA were lysed by passage through a French press device. The French press lysate was partially purified by ammonium sulfate precipitation and the resulting lysate was loaded onto a cibacron blue column and eluted with a NaCl gradient. CheA was finally purified by separation with a G-150 column. The purity and identity of CheA was verified by SDS-PAGE, CnBr digestion and amino acid analysis. Purified material autophosphorylated at the expected molecular weight of CheA.