Ni+-affinity purification of untagged cAMP receptor protein.

The cAMP receptor protein (CRP, also known as CAP) functions as a transcriptional activator at more than 100 different E. coli operons (3). CRP is arguably one of the most well-understood transcriptional activators. At simple CRP-dependent promoters, where CRP is the only activator, transcription activation by CRP involves direct contact with RNA polymerase at two distinct sites. These contacts are made by two patches of surface-exposed residues on CRP known as activating region 1 and activating region 2. Activating region 1 contacts the carboxyl-terminal domain, and activating region 2 contacts the aminoterminal domain of the RNA polymerase α subunit (3). Activating region 1 is located in the DNA-binding domain of CRP, very near the DNA moiety of the CRP-DNA complex, and consists of residues 156-164 (1,3,4,9,15). Activating region 2 is located in the cAMPbinding domain of CRP and consists of residues His19, His21, and Lys101 (3,8, 10,11). While activating region 1 is required for all simple CRP-dependent promoters, activating region 2 is only required when CRP binds adjacent to RNA polymerase (2,3). CRP also activates transcription at many complex promoters that require at least one additional activator protein for maximal expression; however, the mechanism of activation by CRP at such promoters is not as well understood. The structure of CRP has been determined numerous times, in the absence and presence of DNA and of its ligand cAMP (12,13). Purification of CRP is most often accomplished by cAMP affinity chromatography (14), typically using a cAMP-agarose resin [such as adenosine 3′:5′-cyclic monophosphate agarose; Sigma, St. Louis, MO, USA (14)]. While this method utilizes affinity purification and therefore results in purification of CRP in a single chromatography step, the disadvantage of the method is the high cost of the cAMP-agarose resin. CRP has also been previously purified as a His6-CRP fusion protein using immobilized metal-affinity chromatography (IMAC) (5, 7). Here we report the finding that untagged CRP, with no added histidine residues, can be purified to apparent homogeneity using IMAC. The lower cost and widespread use of IMAC resins make this an attractive alternative to cAMP-affinity chromatography. Further, the finding that CRP can be purified by IMAC without the addition of a His6-tag means that any CRP overexpression plasmid can be used with this method and that post-purification protease digestions are not required to obtain untagged CRP. Our finding that untagged CRP could be purified using IMAC arose during the purification of a His6-CRP fusion protein for our studies of the transcription regulation of the E. coli Lrhamnose operons (5,6). We cloned the crp gene into the NdeI and BamHI sites of pET15b (Novagen, Madison, WI, USA) such that it would encode a His6CRP fusion protein with a thrombin cleavage site (pET15b-His6-crp). Strain BL21(DE3) carrying pET15bHis6-crp was grown in 50 mL TY broth (8 g tryptone, 5 g yeast extract, and 85 mM NaCl) with 200 μg/mL ampicillin to an A600 of 0.4–0.6, 1 mM IPTG was added, and the cells were allowed to continue growing for an additional 3 h. The cells were harvested, resuspended in 2 mL of buffer containing 20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 5 mM imidazole, and lysed by sonication. The fusion protein was then purified using IMAC with a 3-mL column of Ni+charged Chelex® 20 resin (Sigma). Elutions were performed with an imidazole step gradient, with steps of 60, 100, 200, and 400 mM, with His6-CRP eluting at 400 mM imidazole. Using this method, we obtained CRP that was apparently homogeneous (based on Coomassie Blue®-stained SDS-PAGE gels) with a yield of greater than 4 mg purified CRP from 50 mL E. coli culture (data not shown). To ensure that the His6-tag did not interfere with the activity of CRP, we treated the purified fusion protein with thrombin (20 U/mg CRP, incubated at 4°C for 24 h; Sigma) to remove the His6-tag. We then attempted to purify the cleaved CRP away from the uncleaved His6-CRP fusion protein, again using Ni+-charged Chelex 20 resin. At this step, the cleaved fusion protein was not found in the flow-through as expected, but rather behaved as though it were binding to the resin. To test this hypothesis, we re-cloned the crp gene into pET15b, but this time we used the NcoI and BamHI restriction sites; therefore, the resulting plasmid (pET15b-crp) would express a fully wild-type CRP with no added histidine residues (untagged CRP). We attempted to purify the untagged CRP using the same growth and purification conditions described above. As shown in Figure 1A, the untagged CRP bound to the Ni+-affinity resin and could be eluted with imidazole. Interestingly, the untagged CRP eluted from the column at a lower imidazole concentration than His6-CRP (200 mM imidazole, and on further refinement of the procedure, 150 mM imidazole), suggesting that the absence of the His6tag weakened the binding of CRP to the resin. In spite of this apparently weaker binding by untagged CRP compared with His6-CRP, the flow-through and low imidazole wash fractions were remarkably free of CRP, and the purified Benchmarks