A NEW EXPRESSION VECTOR FOR HIGH-LEVEL PROTEIN-PRODUCTION, ONE-STEP PURIFICATION AND DIRECT ISOTOPIC LABELING OF CALMODULIN-BINDING PEPTIDEFUSION PROTEINS

Calmodulin-binding peptide (CBP), a peptide of 26 amino acids derived from muscle myosin light chain kinase (MLCK), binds to calmodulin with nanomolar affinity. Proteins fused in frame with CBP can be purified from crude E. coli lysates in a single step using calmodulin affinity chromatography (Stofko-Hahn et al., 1992). Because the binding between CBP and calmodulin is calcium-dependent, the fusion protein can be el uted from the resin with virtually any buffer containing EGTA (2 mM) a nd used directly for many applications. To take full advantage of this affinity purification system, we constructed the versatile CBP fusion protein expression vector pCAL-n. The CBP coding sequence was positio ned for fusion at the N-terminus, an advantage that ensures consistent high level synthesis of fusion proteins due to the efficient translat ion of the CBP in E. coli. The production of fusion proteins from pCAL -n is controlled by the tightly regulated T7/lacO promoter. A versatil e multiple cloning site (MCS) was included to facilitate the cloning o f genes of interest. The protein coding sequence for the enzyme c-Jun N-terminal kinase (JNK) was inserted into the MCS of pCAL-n, and the r esulting fusion protein CBP-JNK synthesized in E. coli cells at 15-20 mg/l culture. CBP-JNK was purified to near homogeneity in one step wit h calmodulin affinity resin. Purified CBP-JNK is fully active, and the CBP peptide tag can be removed by cleavage with thrombin. We also sho w that CBP can be efficiently phosphorylated by cAMP-dependent protein kinase. Hence, the purified fusion proteins can be labeled directly w ith [gamma-P-32]ATP and used to probe protein-protein or protein-nucle ic acid interactions.