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Expression and Purification of Recombinant Proteins That Have Native Amino,,,Acid Sequence

One column purification of protein with native amino acid sequence

Denise L. Wyborski * John C. Bauer * Barbara McGowan * Joseph A. Sorge * Peter Vaillancourt
Stratagene Cloning Systems, Inc.

Stratagene has improved the Affinity protein expression and purification system by the addition of the pCAL-n-EK vector. This E. coli cloning and expression vector is designed for consistent, high-level production and one-step purification of expressed proteins. A ligation-independent cloning (LIC) strategy is used to obtain high-efficiency cloning of the desired protein sequence into the pCAL-n-EK vector. LIC creates seamless cloning junctions between the protein coding sequence of interest and the recognition target for the site-specific protease, enterokinase (EK). The EK target sequence is located between the calmodulin-binding peptide (CBP) purification tag and the N terminus of the expressed protein. Because EK cleaves at the C terminus of its recognition target, which is also the N terminus of the inserted polypeptide sequence, cleavage of fusion proteins produced in the pCAL-n-EK vector yields the desired fusion partner free of any extraneous amino acids derived from the fusion tag. Therefore, cleavage results in the production of native protein.

Stratagene's Affinity protein expression and purification system uses the 26-amino-acid CBP sequence as an affinity tag for purifying recombinant proteins from crude cell lysates with a single pass through calmodulin affinity resin.1,2 The CBP purification system is an excellent alternative to other affinity-tag systems because of its gentle binding and elution characteristics. Another advantage of this system is the small size of the CBP affinity tag (4 kDa). In comparison to other larger affinity tags, the CBP tag is less likely to affect the physical characteristics of the protein of interest.

The pCAL-n-EK vector, a new addition to the Affinity system, contains the CBP coding sequence followed by the EK cleavage site. This configuration allows efficient cleavage of all sequences at the N terminus of the polypeptide of interest and produces proteins with native amino acid sequence.3 Protein coding sequences are cloned into the pCAL-n-EK vector by LIC, a high-efficiency cloning method that does not require ligation or restriction enzyme digestion. LIC creates a seamless junction4,5,6 between the EK cleavage site and the protein coding sequence. The protein sequence is efficiently cloned into the pCAL-n-EK vector, and protein expression is induced by the addition of isopropyl-thio-D-galactoside (IPTG). The fusion protein is purified in one step using the CBP affinity tag. If desired, the affinity tag can be removed by proteolytic treatment with Stratagene's recombinant Enterokinase, and the recombinant protein of interest can be recovered free of CBP-containing digestion products and EK.

Design of the pCAL-n-EK Vector

figure 1

The pCAL-n-EK vector (figure 1) is the fourth in a series of CBP affinity-tag vectors released by Stratagene.1 It is derived from the pET-11 vector series and contains the lacIq gene for expression of the Lac repressor protein and the hybrid T7 promoter for controlled expression of the inserted protein coding sequence.7,8 The vector is transformed into specialized E. coli strains, such as Epicurian Coli BL21(DE3), which contains an expression cassette for T7 RNA polymerase that is induced in the presence of IPTG, allowing tight control and high-level, induced expression of the inserted protein coding sequence. The promoter is followed by the 26-amino-acid CBP affinity tag1,2 and the 5-amino-acid EK cleavage target.3 To provide maximal cloning and expression flexibility, Stratagene has refined the LIC method for cloning inserts into the pCAL-n-EK vector such that there are no constraints on the N-terminal amino acid of the protein coding sequence. The majority of proteins that are expressed in yeast and higher eukaryotes have their N-terminal methionines removed during a posttranslational processing event, an event that is often required to obtain a functional protein.9 This cloning flexibility will be an important feature for researchers who are interested in cloning protein domains, where an N-terminal methionine would be unwanted.

LIC of the Protein Coding Sequence

figure 2

Highly efficient, directional cloning of the desired insert into the pCAL-n-EK vector is by LIC, a method that does not require ligation or restriction enzyme digestion (figure 2).4,5,6 The pCAL-n-EK vector is pretreated to create non-complementary, 12 and 13-nucleotide single-stranded tails at the two 5 ends of the vector. These non-complementary tails prevent the vector from recircularizing, virtually eliminating nonrecombinant plasmids. The insert DNA is prepared by PCR amplification with gene-specific primers that include 12 and 13-nucleotide sequences at the 5 ends that are complementary to the pCAL-n-EK vector single-stranded tails, thus allowing for directional cloning. Following PCR amplification, the PCR product is purified and treated with Pfu DNA polymerase in the presence of dATP. In the absence of dTTP, dGTP and dCTP, the 3 to 5-exonuclease activity of Pfu DNA polymerase removes at least 12 and 13 nucleotides at the respective 3' ends of the PCR product. This activity continues until the first adenine is encountered, producing a DNA fragment with 5'-extended single-stranded tails that are complementary to the single-stranded tails of the pCAL-n-EK vector. 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 the desired insert by PCR amplification.

Efficiency of Cloning into the pCAL-n-EK Vector

To test the efficiency of cloning into the LIC-prepared pCAL-n-EK vector, the sequence encoding c-Jun N-terminal kinase (JNK)10 was PCR amplified using gene specific primers containing the 12 and 13-nucleotide sequences complementary to those in the pCALnEK vector. The 1280-bp JNK sequence that resulted from PCR amplification was gel purified, treated with Pfu DNA polymerase at 72C in the presence of dATP and annealed to the pCALnEK vector containing complementary single-stranded tails. The annealing reaction was transformed into Epicurian Coli XL1-Blue supercompetent E. coli cells and spread onto LB plates containing ampicillin. Of the thousands of colonies that resulted from several transformations, 42 were screened by PCR, and 41 (98%) were found to contain the JNK insert. The accuracy of the LIC cloning method was confirmed by sequence analysis of the regions flanking the N terminus and C terminus of the inserted gene.

LIC cloning was also performed with 50 ng of prepared DNA coding for the kanamycin gene annealed to 30 ng of prepared pCAL-n-EK vector DNA. Onetenth of the reaction was transformed into XL1Blue supercompetent cells, resulting in approximately 800 colony forming units (cfu); 100% were ampicillin and kanamycin resistant. The small amount of insert DNA required for LIC cloning can be obtained from only 10 to 15 cycles of PCR amplification with a high-fidelity enzyme, thus minimizing sequence errors generated by PCR. (For Pfu DNA polymerase, 10 rounds of PCR amplification of a 1kb template results in approximately 99% of the PCR products being free of errors.)

No Constraints on Sequence of Insert DNA

The insert DNA is prepared for cloning by treatment with Pfu DNA polymerase in the presence of dATP. The 3 to 5'-exonuclease activity of Pfu DNA polymerase will remove nucleotides until it encounters the first adenine (figure 2), at which point the 5 to 3-polymerase activity of Pfu DNA polymerase will incorporate the supplied dATP into the sequence.

The PCR product of the pCAL-n-EK vector DNA contains an adenine residue following the C terminus of the insertspecific sequence, which provides a stopping point for the exonuclease activity of Pfu DNA polymerase (figure 2). The N terminus of the insert DNA sequence may not contain an adenine residue for several base pairs past the single-stranded complementary region to the pCALnEK vector, causing a single-stranded gap to form upon annealing insert to the pCALnEK vector. In order to test the effect of single-stranded gaps on the efficiency of LIC cloning, primers were constructed that would produce 17-nucleotide and 27-nucleotide gaps upon annealing the JNK PCR product to prepared pCAL-n-EK vector. (In most experimental cases, gaps will be less than 17 nucleotides long.) Of a 20l annealing reaction, 1 l was transformed into XL1Blue supercompetent cells, resulting in an average of 1250 cfu. Nine clones were sequenced, and eight were found to contain perfect sequence: the 17 and 27-nucleotide gaps had been repaired in vivo by E. coli. This result confirms that there are no constraints on the location of the first adenine residue in the sequence of the insert DNA.

Expression and Purification of CBP Affinity-Tagged Fusion Proteins

The CBP affinity-tag system was used for expressing and purifying the JNK fusion protein from clones of the pCAL-n-EK vector containing the JNK insert ((pCAL-n-EK/JNK).). Epicurian Coli BL21(DE3) competent cells, which encode T7 RNA polymerase, were transformed with a pCAL-n-EK/JNK plasmid, and a culture was grown and induced according to a standard protocol.7 Lysates were prepared, incubated with calmodulin affinity resin, applied to a disposable column, washed with CaCl2 and eluted with 2 mM EGTA as described previously.1 Figure 3 shows the induced and uninduced sample, the calmodulin affinity resin flowthrough fraction depleted of the CBPJNK fusion protein (CBPJNK) and the fraction of pure, 52kDa, EGTAeluted CBPJNK.

Figure 3

Enterokinase Cleavage of the CBP Affinity Tag

The pCALnEK vector contains the 5-amino-acid target sequence for the site-specific protease EK to allow removal of the CBP affinity tag following purification of the fusion protein. Cleavage with EK results in recombinant proteins that contain no extraneous amino acids. Stratagene offers purified recombinant Enterokinase, which exhibits high specific activity and is free of contaminating proteases. Each order of Enterokinase is provided with Soybean Trypsin Inhibitor Agarose. (See the accompanying article in this newsletter, pages 2425, for a description of treating the purified JNK fusion protein with EK. This article also presents purification of the mature cleavage product away from free CBP affinity tag, EK and small amounts of uncleaved fusion protein using Stratagene's Calmodulin Affinity Resin and STI Agarose.)

c-Jun N-Terminal Kinase Activity of CBP-JNK

figure 4

Recombinant CBP-JNK, eluted from the calmodulin affinity resin, was used directly in a protein kinase assay to show that it retained protein kinase activity and substrate specificity. In addition, this fusion protein was able to autophosphorylate and phosphorylate its natural substrate, c-Jun N-terminal kinase (figure 4).

Conclusions

The pCALnEK vector and LIC systems are important new tools for producing proteins with native amino acid sequence. Any desired protein coding sequence can be quickly, efficiently and accurately inserted into the pCALnEK vector by LIC, which permits seamless insertion. Protein expression can be controlled by IPTG, and induced protein can be purified to homogeneity using the CBP purification tag and calmodulin affinity resin. If required, the CBP purification tag can be removed using Stratagene's Enterokinase, yielding a protein with native amino acid sequence. Finally, CBP affinity tag and EK can be removed from the purified protein product using Stratagene's Calmodulin Affinity Resin and Soybean Trypsin Inhibitor Agarose (STI Agarose).

Acknowledgments

The authors would like to thank Quinn Lu, Holly Hogrefe and ChaoFeng Zheng for helpful discussions and Diane Beery for her expertise in preparing graphic art.

REFERENCES

  1. Simcox, T.G., Zheng, C.F., Simcox, M.E., and Vaillancourt, P. (1995) Strategies 8: 4043.

  2. StofkoHahn, R.E., Carr, D.W., and Scott, J.D. (1992) FEBS Lett. 302: 274278.

  3. LaVallie, E.R., et al. (1993) J. Biol. Chem. 268: 2331123317.

  4. Aslanidis, C., and de Jong, P.J. (1990) Nucleic Acids Res. 18: 60696074.

  5. Haun, R.S., Serventi, I.M., and Moss, J. (1992) Biotechniques 13: 515518.

  6. Haun, R.S., and Moss, J. (1992) Gene 112: 3743.

  7. Studier, F.W., et al. (1990) Methods Enzymol. 185: 6089.

  8. Weiner, M.P., et al. (1994) Strategies 7: 4143.

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

  10. Derijard, B., et al. (1994) Cell 76: 10251037.


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