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Efficient Cleavage of Fusion Proteins to Yield Native Amino Termini

vailable enzyme preparations that had been purified from native sources. Such enzyme preparations exhibited low specific activity and contamination with other less specific serine proteases, which often resulted in degradation of target proteins.

Recently, the catalytic subunit of bovine EK has been cloned and expressed in E. coli and purified to homogeneity.1, 2 Stratagene's Enterokinase has high specific activity and is virtually free of contaminating proteases. Purified recombinant Enterokinase is highly active over wide ranges in pH (pH 4.5-9.5) and temperature (4-45C) and efficiently cleaves fusion proteins in partially purified protein preparations.2

Cleavage of CBP-JNK with Enterokinase

figure 2

Coding sequence for the protein c-Jun N-terminal kinase (JNK)3 was PCR amplified using a 5'-oligonucleotide primer designed to fuse the 5-amino-acid EK target site to the natural amino terminus of the JNK protein. The PCR product was inserted into the affinity pCALnEK expression vector4 such that the EK target peptide-JNK fusion protein was fused in frame, downstream of the calmodulin-binding peptide (CBP) purification tag. The resulting fusion protein, CBP-JNK, was expressed in Epicurian Coli BL21(DE3) cells and purified from crude cell lysates to apparent homogeneity, as determined by visual inspection of silver-stained PAGE gels after a single pass over calmodulin (CaM) affinity resin4 (data not shown). A series of reactions containing various amounts of recombinant EK was incubated at room temperature for 21 hours, and portions of the reactions were analyzed by SDS-polyacrylamide gel electrophor
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