Seamless insertion and one-step purification from a more versatile vector
Katherine Felts Denise Wyborski John Bauer Peter
Stratagene has improved the Affinity LIC cloning kit with the new pCAL-n-FLAG vector. This new E. coli cloning and expression vector is designed for consistent, high-level production and one-step purification of expressed proteins. Similar to its predecessor pCAL-n-EK, pCAL-n-FLAG has the same features with the addition of the FLAG epitope ll ll as an N-terminal fusion with the protein of interest. The FLAG epitope is useful to those scientists desiring a small antibody recognition site on their protein of interest. This vector will be of particular interest to scientists who wish to use calmodulin affinity-purified proteins to study protein-protein interactions by pull-down immunoprecipitation experiments using eukaryotic cell extracts, or who wish to probe lambda cDNA expression libraries for interacting proteins. In both cases, the presence of endogenous calmodulin-binding proteins, which are ubiquitous in all eukaryotic cell types, may contribute to background. The calmodulin-binding peptide (CBP) purification tag can be removed from the recombinant protein by thrombin digestion. If desired, the FLAG tag can be removed by digestion with enterokinase, leaving protein of native amino acid sequence.
Purification of recombinant proteins has been greatly simplified in recent years due to the availability of expression vectors that allow fusion of the protein coding sequence of interest to short peptide sequences, or larger proteins, enabling the affinity purification of the fusion protein from crude preparations. The 26-amino acid CBP is an attractive fusion tag for purification of proteins from E. coli extracts1 because of its high affinity for immobilized calmodulin (Kd=10-9). CBP fusion proteins are bound to and eluted from calmodulin affinity matrices at neutral pH using gentle buffer conditions. A relatively low concentration of Ca2+ ions is required for binding (0.2 to 2.0 mM CaCl2), and proteins are efficiently eluted upon removal of the calcium with 2 mM EGTA. The CBP tag is small, (4 kDa) and therefore less likely than some of the larger tags [e.g., the 26-kDa glutathione-S-transferase (GST) and 40-kDa maltose-binding protein (MBP) tags] to affect the biological function of the protein of interest. CBP is efficiently translated in E. coli, and CBP fusion proteins are typically expressed to high levels. In addition, the CBP tag is efficiently phosphorylated by protein kinase A and thus, purified CBP fusion proteins can be readily labeled with isotope and used to probe protein-protein interactions.2
Despite the advantages of using affinity tags for protein purification, there are many proteins for which fusion of nonnatural amino acids at the N- or C-termini adversely affect protein function.3 Most fusion vectors include short recognition sequences for site-specific proteases that allow removal of the fusion tag following purification. Many vectors employ target sequences for proteases that cleave internal to the recognition site and thus, leave at least one extraneous amino acid fused to the protein of interest. The use of the protease enterokinase (EK) has the advantage that it cleaves at the C-terminus of its recognition site, thus when positioned upstream of the protein of interest, N-terminal purification tags can be completely removed without leaving any extraneous amino acid residues on the protein of interest. However, most vectors that employ the use of EK for removal of N-terminal purification tags require the use of cloning schemes (e.g., restriction sites) that constrain the codon choice immediately downstream of the EK cleavage site, and thus predetermine the N-terminal amino acid sequence of the protein of interest.
We recently described the CBP fusion vector pCAL-n-EK4 in which the ligation-independent cloning (LIC) method5 is employed such that any amino acid coding sequence can be seamlessly fused immediately downstream of the EK cleavage site, thereby allowing the recovery of proteins from EK cleavage reactions that have native N-terminal amino acid sequence. In addition to the ability to create seamless fusions between the CBP purification tag and the protein coding sequence of interest, cloning by LIC is highly efficient. Cloning efficiencies of 2 x 105 cfu/g vector are routinely achieved using as little as 25 ng PCR-generated insert; thus, enough insert for several cloning reactions may be generated with as little as 10 cycles of PCR amplification.4
There is an inherent drawback to the use of CBP fusions for certain downstream applications for which purified proteins are employed to probe protein-protein interactions. The ubiquitous presence of calmodulin in eukaryotic cells, in addition to the presence of endogenous calmodulin-binding proteins can, under some conditions, contribute to background in such applications. Specifically, immunoaffinity-type pull-down experiments in which CBP-fusions are recovered from crude eukaryotic extracts by immunoprecipitation with calmodulin affinity resin often result in the recovery of calmodulin-binding proteins.
The pCAL-n-FLAG vector (Figure 1) is the fifth in a series of CBP affinity-tag vectors released by Stratagene. It is derived from the pET-11 vector series and contains the lacI q gene for expression of the Lac repressor protein and the hybrid T7 promoter for controlled expression of the inserted protein coding sequence.8 The vector is transformed into specialized E. coli strains, such as Epicurian Coli BL21(DE3), which contain 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 tag followed by a thrombin cleavage site, the FLAG epitope, an enterokinase cleavage site, the multiple cloning site (MCS), and the T7 transcriptional terminator.
Highly efficient, directional cloning of the desired insert into the pCAL-n-FLAG vector is by ligation-independent cloning (LIC), a method that does not require ligation or restriction enzyme digestion (Figure 2, Panel A). The pCAL-n-FLAG vector is pretreated to create noncomplementary, 12- and 13-nucleotide single-stranded tails at the two 5 ends of the vector. These noncomplementary tails prevent the vector from recircularizing. 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-FLAG vector single-stranded tails, thus allowing for directional cloning. Following PCR amplification , the PCR product is purified, then treated with Pfu DNA polymerase 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.
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).
The CBP affinity purification tag was removed by digestion with thrombin. CBP-FLAG-JNK in elution buffer was supplemented with CaCl2 to a final concentration of 6 mM, then digested at room temperature with a catalytic ratio of thrombin: CBP-FLAG-JNK. To evaluate the extent of digestion over time, the reaction was terminated at various time points (0, 1, and 18 hours). The degree of digestion by thrombin was evaluated by performing SDS-PAGE. 20 l of each time point sample was boiled, then electrophoresed on a 12% Tris-glycine acrylamide gel, and then stained with Coomassie Blue (not shown).
To confirm recognition of FLAG-JNK by anti-FLAG M2 antibody, western blot analysis was performed on the thrombin digest time points. 10-l (~1 g) of sample was subjected to SDS-PAGE, as described above. Samples were electroblotted onto a nitrocellulose filter and detected with anti-FLAG M2 antibody using standard procedures (Figure 4).
It has been shown that transcriptional activity of c-Jun is greatly enhanced in response to phosphorylation.7 JNK binds to the c-Jun transactivation domain and phosphorylates Ser-63, Ser-73, and some secondary sites. To demonstrate the utility of a FLAG-fusion protein with the CBP tag removed, we performed an immunoaffinity pull-down of c-Jun from HeLa cell nuclear extract with immobilized FLAG-JNK.
JNK gel was prepared by immobilizing FLAG-JNK on anti-FLAG M2 affinity gel. JNK gel was equilibrated in binding buffer and subsequently incubated with HeLa extract. The affinity gel was pelleted by low-speed centrifugation and washed extensively with binding buffer. The washed gel pellet was boiled in sample buffer then subjected to SDS-PAGE. Duplicate gels were performed. One gel was stained with Coomassie Blue protein stain, the other gel was electroblotted onto a nitrocellulose filter and subjected to western blot analysis with an anti-c-Jun antibody (Transduction Laboratories, Lexington, KY) by standard procedures. As seen in Figure 5, Panel A, Lanes 3 and 4, roughly equivalent amounts of FLAG-JNK are observed on the Coomassie Blue stained gel. In Figure 5, Panel B, an anti-c-Jun-specific band with a gel mobility corresponding to the expected molecular weight is specifically immunoprecipitated from HeLa nuclear extract with the M2-FLAG-JNK gel (Lane 4).
Stratagene has improved the Affinity LIC cloning kit by the addition of a more versatile vector, pCAL-n-FLAG. This vector has the same features as its predecessor pCAL-n-EK, such as seamless insertion by LIC, and one-step purification using the CBP purification tag and calmodulin resin. The vector has been improved by the addition of the FLAG epitope between the CBP purification tag and the protein of interest. This enables the researcher to remove the CBP from the purified protein while retaining an antibody recognition site. This feature facilitates the use of the recombinant protein of interest in protein-protein interaction experiments using eukaryotic cell extracts. Both the CBP and FLAG fusions can be removed from the purified protein by digestion with enterokinase, liberating protein of native amino acid sequence.
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