An Escherichia coli expression vector that allows recovery of proteins with native N-termini from purified calmodulin-binding peptide fusions

We describe a T7-based Escherichia coli expression vector in which protein coding sequence is seamlessly fused to the N-terminal calmodulin-binding pe ptide (CBP) purification tag. We combined the use of the site-specific prot ease enterokinase (EK) and the type IIs restriction enzyme Eam1104 I, which cleave outside their respective (amino acid and nucleotide) target sequenc es, such that any amino acid sequence may be fused directly C-terminal to t he EK cleavage site without codon constraints conferred by the cloning meth od. PCR products are cloned using ligation-dependent or ligation-independen t methods with high cloning efficiencies (>10(6) cfu/mu g vector), allowing production of insert quantities sufficient for several cloning experiments with a limited number of PCR cycles, resulting in a significant time-savin gs and reduced likelihood of accumulating PCR-derived mutations. CBP fusion proteins are expressed to high levels when the CBP peptide is positioned a t the N-terminus. CBP binds to calmodulin with nanomolar affinity, and fusi on proteins are purified to near homogeneity from crude extracts with one p ass through calmodulin affinity resin using gentle binding and elution cond itions. We show high efficiency seamless cloning of three inserts into the pCAL-n-EK vector, including one encoding the protein c-Jun N-terminal kinas e (JNK). CBP-EK-JNK fusion protein was synthesized to 10-20 mg/liter cultur e and purified to near homogeneity in one step wit calmodulin affinity resi n. The fusion tag was efficiently removed wit EK to yield active JNK wit na tive N-terminal amino acid sequence. (C) 1999 Academic Press.