OVER-EXPRESSION, PURIFICATION AND PHOSPHORYLATION OF THE BACILLUS-SUBTILIS CHEA PROTEIN

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