3, 417-420



Preparation and Properties of an Affinity Support for Purification of Cyclic AMP Receptor Protein from Escherichia co/i John E. T. Corrie,*,l





and Rodney

W. King*

*National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 IAA, United Chimica delle Sostanze Naturale, Via Domenico Montessano 49, Napoli, Italy; and $Department Brunel University, Uxbridge, Middlesex UB8 3PH, United Kingdom



21, 1992;

and in revised



23, 1992

Reaction of cyclic AMP with l,l’-carbonyldiimidazole produces an intermediate which reacts with primary amines to provide a stable 2’-0-carbamyl derivative. This chemistry has been used to tether cyclic AMP to a Sepharose gel. The resulting affinity support has been used to effect a simple, nondenaturing purification of cyclic AMP receptor protein from crude cell extracts. 0 1992 Academic Press, Inc.

The cyclic AMP receptor protein of Escherichia coli binds to DNA in the presence of CAMP and regulates expression of a number of genes, including those encoding catabolic enzymes for several nonglucose sugars and its own structural gene (l-4). Purification of cyclic AMP receptor protein from broken cell preparations has hitherto required three consecutive column fractionations [ (5) and references therein] and in order to facilitate isolation of both the native protein and modified forms thereof for use in NMR studies (6,7), we examined the use of affinity chromatography as a more convenient means of purification. Since CAMP is the natural ligand for the protein, we proposed tethering the cyclic nucleotide to a solid support such that the ability to bind to the protein is retained, and data from a previous study on the binding of modified cyclic nucleotides to cyclic AMP receptor protein (5) indicated that linkage via the 2’-hydroxyl group should be an appropriate strategy. Preliminary investigation of 2’-0-succinyl-CAMP (8) suggested that the ester linkage was unacceptably labile and so we turned our attention to the more stable 2’-0-carbamyl derivatives. To our knowledge such derivatives of CAMP have not been described previously and in order to validate the chemistry we first examined the synthesisandcharacterizationof2’-0-(N-(2-acetamino1 To whom



1046.5928192 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form

be addressed.

Kingdom; TDipartimento di of Biology and Biochemistry,

ethyl)carbamyl)cAMP (1). These results and their extension to the preparation of the CAMP affinity column are described below. MATERIALS




CAMP (free acid), 4-morpholine-N,N’-dicyclohexylcarboxamidine, and DEAE-cellulose were from Sigma; l,l’-carbonyldiimidazole from Lancaster Synthesis was used only if its melting point was found to be above 114°C. AH-Sepharose 4B was from Pharmacia; all other reagents, including anhydrous dimethylformamide, were from Aldrich. ‘H and 31P NMR spectra in D,O solution were obtained, respectively, on Bruker AM 500 and WM 200 instruments. The fast atom bombardment mass spectrum was obtained on a VG 70-250SE instrument with the sample in a glycerol-thioglycerol matrix. 2’-O-(N-(2-Acetaminoethyl)carbamyl)cAMP


Tri-n-butylamine (0.36 ml, 1.5 mmol) was added to a suspension of CAMP (165 mg, 0.5 mmol) in methanol (5 ml), the mixture was refluxed until it became homogeneous, and the solvent was evaporated under reduced pressure. The residue was dissolved in anhydrous dimethylformamide (5 ml) and NJ/‘-dicyclohexyl-4-morpholinecarboxamidine (147 mg, 0.5 mmol) was added. The mixture was warmed gently until it became homogeneous, the solvent was evaporated under reduced pressure, and the residue was dried by repeated vacuum evaporation of anhydrous dimethylformamide and then kept under vacuum overnight over P,O,. The dried N,N’-dicyclohexyl-4-morpholinecarboxamidinium CAMP salt (0.5 mmol) was dissolved in anhydrous dimethylformamide (5 ml) and treated with 417

Inc. reserved.





Hz). Under the same conditions, at 6 -1.68. Linkage of CAMP to AH-Sepharose




FIG. 1. Synthesis agents are: (i) AcNHCH,CH,NH,;

of 2’-0-carbamyl derivatives of cyclic l,l’-carbonyldiimidazole/dimethylformamide; (iii) AH-Sepharose 4B/aq.DMF.



l,l’-carbonyldiimidazole (400 mg, 2.5 mmol). The solution was stirred at room temperature for 2 h and then methanol (90 ~1, 2 mmol) was added to quench excess l,l’-carbonyldiimidazole, followed by N-acetylethylenediamine (255 mg, 2.5 mmol) (Fig. 1). The progress of the reaction was followed by reverse-phase HPLC, using a Merck Lichrocart RP-8 column (250 X 4 mm) and 10 mM potassium phosphate, pH 5.451methanol (95/5) as mobile phase, at a flow rate of 1.5 ml/min. Retention times for CAMP and the acetamino compound 1 were 4.9 and 10.7 min, respectively. Formation of compound 1 reached a maximum after 2 h when the yield before purification, estimated from the HPLC peak height, was 75%. The solvent was evaporated under reduced pressure and the residue was dissolved in deionized water (350 ml) and applied to a column of DEAE-cellulose (35 X 2.5 cm) equilibrated with 10 mM triethylammonium bicarbonate buffer, pH 7.8. The column was eluted at 60 ml/h with a linear gradient formed from 10 and 200 mM triethylammonium bicarbonate (each 1000 ml) and 12-ml fractions were collected. Fractions 50-70 showed uv absorbance at 258 nm and were assayed by analytical HPLC as above. Most fractions contained varying ratios of CAMP and the derivative 1, but fractions 63 and 64 contained only compound 1: Fast atom bombardment mass spectrum (negative ion) found, m/z 456; calculated for C,,H,,N,O,P, m/z 456; ‘H NMR (referenced to HDO signal at 6 4.85) 8.33 (lH, s, H-8), 8.31 (lH, s, H-2), 6.39 (lH, s, H-l’), 5.63 (lH, d, J2,,3, = 5.7 Hz, H-2’), 5.11 (lH, m, IV, = 18 Hz, H-3’), 4.62 (lH, ddd, J4,,5’ = 3 Hz, J5’,5w= 8 Hz, J5s,p= 20 Hz, H-5’), 4.40-4.50 (2H, m, H-4’,5”), 3.35-3.45 (4H, m, -CH,CH,-), 2.08 (3H, s, CH,CO); 31P NMR (referenced to 85% H,PO, at 6 0) -2.26 (d, JHs,,p = 20

CAMP gave a signal


A solution of N,N’-dicyclohexyl-4-morpholinecarboxamidinium CAMP salt (0.1 mmol; prepared as described above) was dissolved in anhydrous dimethylformamide (1 ml) and treated with l,l’-carbonyldiimidazole (80 mg, 0.5 mmol). After 2 h at room temperature, the solution was treated with methanol (18 ~1,0.4 mmol) and the solvents were evaporated in uacuo. The residue was redissolved in anhydrous dimethylformamide (0.5 ml). Meanwhile, AH-Sepharose 4B (0.5 g) was reconstituted according to the manufacturer’s protocol and finaily suspended in distilled water. After settling under gravity the supernatant was decanted from the gel (swollen volume, 2 ml), which was combined with the dimethylformamide solution of activated CAMP, and the mixture was kept at room temperature on an endover-end mixer for 72 h. The gel was then washed exhaustively with water and methanol, resuspended in water, and stored at 4°C. To quantitate the extent of derivatization of the gel, the concentration of CAMP recovered in the combined water and methanol washings was determined from its uv absorption (9) at 260 nm (t = 15,400 M-’ cm-‘). From this figure and the measured volume of the washings, the total of CAMP not bound to the gel can be determined and hence the amount attached to the gel can be calculated. Gel performance was monitored in two stages. A small-scale (1.5 ml) column was poured and extensively washed with 20 mM phosphate buffer, pH 7.5, containing 1 mM EDTA, 7 mM mercaptoethanol, and 200 InM KCI. The load was a 30-50% ammonium sulfate fraction of crude cell extract, dialyzed extensively against the column buffer. An aliquot (2 ml) of the load was applied to the column, which was developed with buffer solution and collected in fractions (1.5 ml). The fractions were examined for optical density at 278 nm, for CAMP binding activity (5), and on SDS-PAGE. Results are shown in Fig. 2. It is clear from these results that although cyclic AMP receptor protein does not bind tightly to the column, it is retarded, and even under these conditions of high overload, some purification is achieved. An exactly similar column, poured from unreacted gel, showed no retardation of the CAMP binding activity relative to the optical density. A large-scale column was prepared using 40 ml of the CAMP-substituted gel. The load for this column was 8 ml of the dialyzed ammonium sulfate fraction. Development of the column with the same phosphate buffer solution and collecting 5ml fractions gave the result shown in Fig. 3. The CAMP binding activity is well separated from the optical density and most importantly the recovery of CAMP binding activity is greater than 85%.






g = P P


a 5 m


B " 5. z Z



s ii = 4



5 Fraction





FIG. 2. Small-scale test of CAMP affinity gel: 1.5 ml ofthe gel equilibrated with 20 mM phosphate buffer, pH 7.5, containing 1 mM EDTA, 7 mM mercaptethanol, and 200 mM KC1 was poured into a column; 2 ml of a dialyzed 30-50% ammonium sulfate fraction of crude cell extract was applied and eluted with the same buffer solution; and 1.5ml samples were collected and the above elution profile of uv absorbance and CAMP binding activity was obtained. Inset shows SDSPAGE of the fractions numbered. The darkest band in fractions 4 and 5 corresponds to CRP.

This semipurified material is at the correct pH and salt concentration to be used as the load for a final phosphocellulose column purification (5). RESULTS




of dicyclohexyl-4-morpholinecarboxamidinium CAMP salt and excess ethylenediamine in dimethylformamide at room temperature showed no change in the concentration of CAMP over at least 4 days. We note that for preparation of the CAMP salt it was found necessary to use twice the molar ratio of tri-n-butylamine previously described (14) in order to effect the initial solution in methanol. When the CAMP salt was treated first with l,l’carbonyldiimidazole and then N-acetylethylenediamine (see Fig. l), reverse-phase HPLC showed the formation of a product more hydrophobic than CAMP. Surprisingly, fractionation of the reaction mixture on DEAEcellulose yielded a few fractions in which this product was uncontaminated by CAMP, and this material was adequate for spectral characterization. We note that preparative reverse-phase HPLC would be a more effective means of isolating the full yield of this reaction product. Analysis of this product by ‘H NMR confirmed that the reaction product was the 2’-0-carbamyl derivative 1. The ribose region of the spectrum was very similar to that previously reported for a 2’-0-acylated derivative, namely N,2’-dibutyryl-CAMP (15). Thus the downfield shift of 2’-H confirms the presence of a 2’-0-acyl substitutuent, while properties such as the very small 1’,2’ coupling constant and the large 5-H, 31P coupling constant confirm the integrity of the cyclic phosphate. Since only a single acetamino methyl resonance was present, associated with the substituted 2’-0-carbamyl group, the possibility that a phosphoramidate had also been formed was excluded, and this conclusion was sup-


l,l’-Carbonyldiimidazole has been used previously to prepare the 2’,3’-cyclic carbonate of nucleotides (10) and the ADP derivative has been reacted with a primary amine to form a base-stable carbamyl derivative (11). The same chemistry has been used to prepare an ADP affinity column (12). However, with these noncyclic nucleotides, carbonyldiimidazole also activates the terminal phosphate group and hence treatment with a primary amine produces a phosphoramidate, which necessitates a subsequent acid-catalyzed cleavage of the P-N bond to regenerate the phosphate (11). Since the reaction has not been applied to cyclic nucleotides, we were concerned initially to establish several features: first, whether a primary amine would cleave the cyclic phosphate, second, whether sequential treatment with l,l’carbonyldiimidazole and a primary amine would generate the desired 2’-0-carbamyl derivative, and third, whether any reaction, probably to form a cyclic phosphoramidate, would occur at the cyclic phosphate. Derivatives of the latter type can be prepared by activation of a 3’,5’-cyclic nucleotide with other reagents [ (13) and references therein]. The first question was readily answered when anionexchange HPLC analysis (data not shown) of a solution










FIG. 3. Production scale test of CAMP affinity gel: 40 ml of the gel equilibrated with the 20 mM phosphate starting buffer was poured into a column; 8 ml of a dialyzed 30-50% ammonium sulfate fraction of crude cell extract was applied and the column developed with the same buffer solution; and 5-ml fractions were collected and the above elution profile of uv absorbance and CAMP binding activity was obtained. Inset shows SDS-PAGE of the fractions numbered. The darkest band in fraction 15 corresponds to cyclic AMP receptor protein.




TABLE Purification Total binding

Step Supernatant from disruption of 35 g of cells Resuspended and dialyzed 30-50% saturation ammonium sulfate precipitate Pooled affinity column effluent Pooled phosphocellulose column effluent n One




the binding

of 1 pmol



CAMP units”




Total optical density at 280 nm (sample volume x O.D. 1 cm)

2.8 X lo5


2.1 x lo5


1.3 x lo5 1.2 x lo5 of CAMP


E. coli


ported by the 31P NMR resonance, which was only slightly shifted from the value for CAMP itself. Finally, the molecular ion observed in the negative-ion FAB mass spectrum was consistent with the empirical formula of compound 1. The binding constant of 1 to cyclic AMP receptor protein was determined by competition with radioactively labeled CAMP as described in (5). The dissociation constant, Kd = 2.5 + 0.05 X lop4 M, is almost two orders of magnitude weaker than that for CAMP. With the chemistry of this model 2’-0-carbamoyl derivative securely established, it was possible to proceed to the preparation of the affinity support. This was readily achieved by using the primary amino groups on AH-Sepharose 4B to react with the carbonyldiimidazole-activated CAMP. Thus the swollen gel was slurried with an approximately lo-fold excess of activated CAMP, and the decrease in uv absorbance of the supernatant at the end of the reaction time was consistent with the capture of approximately 10% of the total CAMP by the gel. While this value may be subject to considerable error, it suggests that most of the available amino groups on the gel had been derivatized. We have not attempted more accurately to quantitate the CAMP loading of the gel, although this could be achieved if desired by incorporating a small amount of radiolabeled CAMP in the activation and coupling procedure. The purification of cyclic AMP receptor protein from bacterial extracts has until now presented us with some problems associated with steps involving lowered pH, where irreversible or only partially reversible precipitation of the CAMP binding activity has led to major losses of protein. This is particularly acute when, for NMR investigations, the protein contains specifically incorporated fluorinated or isotopically labeled amino acids, or has been perdeuterated or grown in a 15N medium. The use of this new column material enables purification through a two-column procedure, with no change in the pH of the supporting buffer and a consequent reduction in losses during the procedure. Table 1 shows typical results for a purification run using the affinity column.



8.1 4.3 standard

Purification (fold)




185 321



The methodology described here may have application in the preparation of other cyclic nucleotide affinity supports. In addition it could be used to synthesize other stable conjugates of cyclic nucleotides, for example, with fluorophores or radioactively labeled species. Such derivatives may have applications in immunoassay or other biochemical techniques. ACKNOWLEDGMENT We are ment.


to Mr.

S. Howell






REFERENCES 1. de Crombrugghe, B., and Pastan, I. (1978) in The Operon (Miller, J., and Reznikoff, W., Eds.), pp. 3033324, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 2. de Crombrugghe, B., Busby, B., and But, H. (1984) in Biological Regulation and Development (Goldberger, R. F., and Yamamoto, K. R., Eds.), Vol. 3B, pp. 129-167, Plenum, New York. 3. Aiba, H. (1983) Cell 32, 141-149.

4. Schultz, S. C., Shields, G. C., and Steitz, T. A. (1991) Science 253, 1001-1007. 5. Donoso-Pardo, J. L., Turner, P. C., and King, R. W. (1987) Eur. J. Biochem. 168,687-694. 6. Sixl, F., King, R. W., Bracken, M., and Feeney, J. (1990) Biochem. J. 266,545-552. 7. Hinds,

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R. M., Ling, N. S., Morrell, S. A., and Lipton, Biochem. Biophys. 62, 253-264.

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A. D., and


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11. Cremo, C. R., Neuron, J. M., and Yount, R. G. (1990) try 29,3309-3319. 12. Braxton, S., and Yount, R. G. (1988) Biophys. J. 53, 13.

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J. Am.

Preparation and properties of an affinity support for purification of cyclic AMP receptor protein from Escherichia coli.

Reaction of cyclic AMP with 1,1'-carbonyldiimidazole produces an intermediate which reacts with primary amines to provide a stable 2'-O-carbamyl deriv...
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