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Efficient Cleavage of Fusion Proteins to Yield Native Amino Termini

Recombinant Enterokinase for highly specific digestion of purified proteins or proteins in crude extracts

Ed Marsh * Johanna Claycomb * ChaoFeng Zheng * Denise Wyborski * John C. Bauer * Joseph A. Sorge * A.K. Bernardino * Peter Vaillancourt
Stratagene Cloning Systems, Inc.

Stratagene introduces recombinant Enterokinase, an efficient, sitespecific protease that cleaves at the carboxy terminus of its recognition target. Cleavage of fusion proteins that contain the enterokinase (EK) recognition sequence positioned between an Nterminal purification tag and the Cterminal polypeptide of interest results in Cterminal fusion partners that have no extraneous amino acids derived from the cleavage site. Thus, recombinant proteins with native amino acid sequence are produced. Stratagene's purified Enterokinase is free of contaminating proteases, functions efficiently over a wide range of reaction conditions and is provided with Soybean Trypsin Inhibitor Agarose (STI Agarose) for rapid and quantitative removal of EK from digests.

figure 1

Site-specific proteases and their cognate target amino acid sequences are often used to remove the fusion protein tags that facilitate enhanced expression, solubility, secretion or purification of the fusion protein. The serine protease enterokinase (EK) is particularly attractive because it cleaves after the C-terminal amino acid of its recognition site, (aspartic acid)4lysine. This cleavage produces fusion proteins that have native amino termini following removal of N-terminal tags by cleaving with EK (figure 1). In the past, the use of EK for fusion protein cleavage was hampered by the generally poor quality of the commercially a 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 esis ((SDS-PAGE).). As the results in figure 2 indicate, after a 21-hour incubation, all of the fusion protein is cleaved in the absence of any detectable nonspecific proteolytic activity.

Figure 3

Following purification of fusion proteins and proteolytic removal of the affinity peptide tags, recombinant EK reactions contain a mixture of the protein of interest, the free affinity tag, recombinant EK and, occasionally, small amounts of uncleaved fusion protein. Recovery of the mature cleavage product from this mixture is often desired. Figure 3 (Panel A) shows the results of a simple and efficient one-step protocol for removing both recombinant EK and products that contain the CBP affinity tag. In the first experiment, the fusion protein was briefly digested with recombinant EK to yield a mixture of uncleaved fusion protein, mature JNK cleavage product and the free CBP tag (lane 2). The reaction was absorbed for 30 minutes with a mixed slurry of CaM affinity resin and STI Agarose (the latter is for the removal of recombinant EK, not visible on the gel). In this experiment, all of the detectable fusion protein and CBP were bound to the resin (lane 4), while the vast majority of the JNK cleavage product remained in the unbound fraction (lane 3). In a separate experiment, (Panel B) a reaction containing an excess of recombinant EK (200 ng) was split and absorbed with either CaM affinity resin alone (STI, +CaM) or with a mixed slurry of CaM affinity resin and STI Agarose (+STI, +CaM), and the unbound material was assayed for the presence of recombinant EK activity using a fluorescent peptide substrate (see legend to Figure 3 ). As the results in panel B show, the recombinant EK was quantitatively removed when the STI Agarose was added to the absorption step.

Conclusions

Stratagene's pure, high-specific-activity, recombinant EK allows efficient, specific cleavage of fusion proteins over a wide range of conditions. Recombinant EK can be used in conjunction with the Affinity and ESP protein expression and purification systems and, in particular, with the new E. coli fusion vector, pCALnEK, to produce unmodified cloned proteins in high yield. With the introduction of STI Agarose, cleaved recombinant proteins with native amino acid sequence can be efficiently recovered from recombinant EK digests without buffer changes or long incubation times.

REFERENCES
  1. LaVallie, E.R., et al. (1983) J. Biol. Chem. 268: 2331123317.

  2. CollinsRacie, L.A., et al. (1995) Biotechnology 13: 982987.

  3. Zheng, C.F., and Simcox, T.G. (1996) Strategies 9: 2021.

  4. Wyborski, D.L., Bauer, J,C., McGowan, B., Sorge, J.A. and Vaillancourt, P. (1997) Strategies 10: 1517.

  5. Hirel, P.H., et al. (1989) Proc. Nat. Acad. Sci. USA 86: 82478251.


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