( e in the presence of dATP. The vector ...)High-Level Protein Expression, One-Column Purification, and FLAG,,,Epitope Tagging in E. coli

e in the presence of dATP. The vector and insert DNA are combined, allowed to anneal at room temperature, and transformed into highly competent bacterial host cells. The resultant colonies can then be screened for insert by standard methods.

The 1280-bp gene encoding c-Jun N-terminal kinase (JNK) was PCR amplified with gene-specific primers containing LIC ends. The JNK PCR fragment was prepared for LIC using the Stratagene Affinity LIC cloning and protein purification kit. The LIC-ready pCAL-n-FLAG and JNK insert were combined according to the kit instructions and transformed into XL1-Blue supercompetent cells. Positive clones were subsequently confirmed by DNA sequence analysis.

Expression and Purification of CBP-FLAG-JNK

Figure 3

The pCAL-n-FLAG-JNK construct was transformed into BL21 (DE3) cells and JNK expression was induced with IPTG, according to standard protocols.⁸ To confirm protein induction, SDS-PAGE was performed on the uninduced and induced culture samples. 20 l of culture was added to 20 l of 2X reducing sample buffer and fractionated on a 4 to 20% acrylamide Tris-glycine gel. The gel was stained with Coomassie Blue and analyzed for protein induction (Figure 3, Panel A). CBP-FLAG-JNK was then purified over calmodulin resin. The cleared lysate was incubated with calmodulin affinity resin for 2.5 hours. The slurry was transferred to a disposable column and washed extensively. CBP-FLAG-JNK was eluted with elution buffer containing 2 mM EGTA. (Figure 3, Panel B).

Removal of CBP Tag by Thrombin Digestion and Anti-FLAG Western Blot of FLAG-JNK

The CBP affinity purification tag was removed by digestion with thrombin. CBP-FLAG-JNK in elution buffer
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