Bioconjugate Chem. 1996, 7, 126−130
126
On-Off Switching of Enzymatic Reaction by Recombinant Calmodulin on a Solid-Phase Matrix Eiry Kobatake,† Koichi Mitomo,† Tetsuya Haruyama,† Masuo Aizawa,*,† Glenn Y. Deng,‡ and Seishi Kato‡ Department of Bioengineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226, Japan, and Sagami Chemical Research Center, 4-4-1 Nishi-ohnuma, Sagamihara, Kanagawa 229, Japan. Received May 30, 1995X
A fusion protein consisting of human calmodulin (CaM) and glutathione S-transferase (GST) was produced by gene fusion. The fusion protein was overexpressed in Escherichia coli as a soluble form and purified with one-step affinity chromatography using glutathione-Sepharose. The protein had the modulating activity of CaM and the binding capability to glutathione of GST. Phosphodiesterase, which is a CaM dependent enzyme, was activated by the fusion protein, with the Ca2+ level equal to the level equivalent to a native CaM. Furthermore, CaM could be immobilized on a solid-phase matrix through the use of GST moiety while its modulating activity was retained. Phosphodiesterase activity was switched on and off by the immobilized CaM with or without Ca2+, and repeated use of CaM was demonstrated.
INTRODUCTION
Advances in protein engineering have led us to design a variety of fusion proteins by assembling several functions of proteins. For example, two enzymes, which catalyze sequential reactions, have been fused to perform at an enhanced reaction rate due to the proximity of the enzymes (1, 2). There is an advantage in facilitating the sequential catalysis by two or more enzymes for the biotechnological production process. Another approach for utilizing the fusion protein is using a binding protein for biological analysis and protein purification. Some reports have demonstrated the fusion protein as a reagent for enzyme immunoassay (3, 4). In previous reports, we have shown an applicability of the fusion protein between marker enzymes and protein A, which binds to IgG molecules, in enzyme immunoassay (5, 6). The gene fusion systems for the expression and purification of recombinant proteins have been studied extensively (7-9). In these systems, the structural gene of interest is connected in frame to a gene encoding a protein which binds to a ligand by affinity, and the resulting protein can be readily purified only through the use of affinity chromatography. Another expected advantage of the fusion protein containing a binding protein is the capability of immobilizing the functional protein on a matrix. Through fusion of a binding protein to a functional protein such as an enzyme, we expected that the functional protein, while controlling the orientation of an active site in the molecule, can be assembled. Such hybridization of proteins by gene fusion has been attracting attention as a useful technique for immobilization and assembly of functional proteins. Immobilization of biological materials offers several advantages, for instance, stabilization of the intrinsically unstable materials and increasing the efficiency of the reaction by assembling the functional molecules. More* Author to whom correspondence should be addressed. Telephone: +81-45-924-5759. Fax: +81-45-924-5779. † Tokyo Institute of Technology. ‡ Sagami Chemical Research Center. X Abstract published in Advance ACS Abstracts, December 15, 1995.
1043-1802/96/2907-0126$12.00/0
over, it is possible to use rather expensive biological materials continuously and repeatedly. Immobilizing techniques have already been utilized for immobilization of enzymes or antibodies for the manufacturing of biosensors and bioreactors (10). Besides, the ligands immobilized on a matrix are very useful tools for purification of materials which have affinities for ligands as described above. Calmodulin (CaM)1 undergoes remarkable conformational change when it binds Ca2+ (11, 12), which results in the modulation of many important biological reactions (13, 14). This regulation is one of the key steps in a variety of cellular processes controlled by Ca2+. By immobilizing CaM while retaining its activity to a solidphase matrix, we found that it is a feasibile to control the conformational change of CaM and the activity of CaM dependent enzymes by Ca2+, continuously and reversibly. One successful approach for such immobilization of CaM has been demonstrated by chemical coupling. CaM immobilized on Sepharose has been used for the affinity purification of CaM dependent proteins (15-17). However, in these cases, CaM molecules are immobilized on a matrix with random orientation. For the exhibition of the efficient enzymatic reaction, it is important not only to bind the target proteins for the purification but also to immobilize CaM molecule while controlling their orientation. For construction of such a system, we prepared a monolayer membrane of CaM modified with lipid at the air-water interface by the Langmuir-Blogett method and showed that the conjugated protein film activates the CaM dependent enzyme, depending on Ca2+ concentration, in our previous work (18). In this study, we prepared a recombinant CaMglutathione S-transferase fusion protein for the purpose of immobilizing a modulating protein while controlling its orientation on a solid-phase matrix. It is possible to immobilize CaM on a glutathione-immobilized matrix through the use of glutathione S-transferase (GST), 1 Abbreviations: CaM, calmodulin; GST, glutathione S-transferase; IPTG, isopropyl β-D-thiogalactoside; PDE, 3′,5′-cyclicnucleotide phosphodiesterase; cAMP, cyclic 3′,5′-monophosphate; ADA, adenosine deaminase; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.
© 1996 American Chemical Society
On−Off Switching of Enzymatic Reaction
Figure 1. Map of the plasmid pGEX-CaM: GST, structural gene for glutathione S-transferase; CaM, structural gene for CaM; AmpR, ampicillin resistance gene; lacIq, lac repressor gene; and Ptac, tac promoter. Arrows indicate the direction of transcription. The sequences of nucleotides and amino acids in the junction region between GST and CaM are also shown. Factor X indicates the factor Xa site in the resulting fusion protein.
because GST can bind to matrix-bound glutathione. Furthermore, on-off switching of phosphodiesterase activity by CaM with or without Ca2+ was investigated. This approach is expected to apply not only to a bioreactor system for CaM dependent enzymes but also to the delivery of drugs such as calcitonin and parathyroid hormone, which are responsible for the regulation of Ca2+ concentration in serum. EXPERIMENTAL PROCEDURES
Materials. Plasmid pGEX-3X was purchased from Pharmacia (Uppsala, Sweden). Restriction enzyme, XmnI, was obtained from New England Biolabs (Beverly, MA), and other restriction enzymes, ligase, T4 DNA polymerase, and factor Xa, were purchased from Takara Shuzo (Shiga, Japan). 3′,5′-Cyclic-nucleotide phosphodiesterase was from Sigma (St. Louis, MO). Glutathione-Sepharose beads were from Pharmacia, and all other chemicals were of analytical grade. Construction of Plasmid. The GST-CaM gene fusion vector, pGEX-CaM, was constructed as follows. The coding region of human CaM cDNA (19) was amplified by polymerase chain reaction using synthetic oligonucleotides, 5′-AGGGAAGGATTTCAGCTGACCAACTGACTGAAG and 5′-CAGGTCGACTCACTTTGCTGTCATCATTTG. The amplified fragment was digested with XmnI and SalI, and subsequently, both terminal ends were blunted with T4 DNA polymerase. The isolating gene fragment of CaM was ligated to the SmaI site of the pGEX-3X with T4 DNA ligase. In the resulting plasmid, the gene of CaM was fused to the gene of GST in frame. The constructing plasmid is schematically drawn in Figure 1. Expression and Purification of the Fusion Protein. The Escherichia coli strain JM105 cells transformed with plasmid, pGEX-CaM, were cultured in 400 mL of LB medium containing ampicillin (50 mg mL-1)
Bioconjugate Chem., Vol. 7, No. 1, 1996 127
at 37 °C with shaking. In the midexponential phase, isopropyl β-D-thiogalactoside (IPTG) was added to a final concentration of 0.1 mM and the mixture cultured for another 3 h. The bacterial cells were harvested by centrifugation at 4000g for 10 min at 4 °C, and the cell pellet was washed twice with cold STE buffer [0.1 M NaCl, 10 mM Tris-HCl, and 1 mM ethylenediaminetetracetic acid (EDTA) (pH 7.8)] and resuspended in 10 mL of the same buffer containing 1% Triton-X100. The cell suspension was sonicated for 30 min and centrifugated at 5000g for 10 min to remove the cell debris. The fusion protein was purified through the use of glutathioneSepharose beads as described by the manufacturer (Pharmacia). The resulting supernatant was mixed with an equal volume of glutathione-Sepharose beads and incubated for 30 min at room temperature. The beads were washed three times with PBS [150 mM NaCl, 16 mM Na2HPO4, and 4 mM NaH2PO4 (pH 7.3)] and then mixed with elution buffer [50 mM Tris-HCl (pH 8.0) containing 50 mM glutathione]. The protein molecules bound to the beads were released competitively from the beads with the elution buffer. The solution containing the fusion protein was obtained from the supernatant by centrifugation. Isolation of the CaM Moiety. There is a recognition site of factor Xa, which is a site specific protease (20), between GST and CaM (Figure 1). For cutting off the protein by factor Xa, the concentration of Ca2+ in the protein solution was adjusted to 1 mM with CaCl2. Then factor Xa was added at a 1:100 (factor Xa:fusion protein) molar ratio and the mixture incubated for 2 h at 25 °C. When the reaction mixture was applied to glutathioneSepharose beads again, the GST moiety bound to the beads and only the CaM moiety remained in the solution. Activity Measurement of PDE. The activity of CaM was estimated as an activator for 3′,5′-cyclic-nucleotide phosphodiesterase (PDE), which is one of the CaM dependent enzymes (21). The measurement was followed by the method of Schiefer (22). PDE is activated in the presence of CaM which binds Ca2+ and catalyzes the hydrolysis reaction of cyclic adenosine 3′,5′-monophosphate (cAMP) to adenosine 5′-monophosphate (AMP). Then the AMP formed from cAMP in the reaction is dephosphorylated to adenosine in the presence of alkaline phosphatase. Subsequently, adenosine is hydrolyzed to inosine in the presence of adenosine deaminase (ADA). The PDE activity was determined by monitoring the decrease in absorbance at 265 nm which accompanies consumption of cAMP. CaM solution (10 µL) was mixed thoroughly with a glycylglycine buffer (100 mM, pH 7.5) containing MgSO4 (1.2 mM), CaCl2 (2.1 mM), ADA (4 ku/L), alkaline phosphatase (30 ku/L), and PDE (3u/L) and the mixture incubated for 10 min at 30 °C. Finally, a solution of cAMP was added to a final concentration of 87 mM, and the final volume of the assay mixture was 1.0 mL. Immobilization of CaM on a Mixture. The fusion protein was immobilized on glutathione-Sepharose beads which were used in the purification process. Cell lysate containing the fusion protein was mixed with glutathione-Sepharose and incubated for 30 min. The beads were washed well with PBS to remove proteins which adsorbed nonspecifically on a matrix. The resulting beads were directly used as the fusion proteinimmobilized solid-phase matrix. RESULTS AND DISCUSSION
Expression of the Fusion Protein. The constructing plasmid, pGEX-CaM, possessed the fusion gene of GST and CaM under the control of the tac promoter
128 Bioconjugate Chem., Vol. 7, No. 1, 1996
Figure 2. SDS-PAGE analysis of the fusion protein. E. coli JM105 cells, with or without plasmid pGEX-CaM, were lysed with sonication, and soluble proteins were analyzed: lane 1, standard samples (molecular mass is indicated on the left); lane 2, JM105/pGEX-CaM cell lysate induced by IPTG; lane 3, JM105/pGEX-CaM cell lysate (noninduced); and lane 4, JM105 cell lysate.
(Figure 1). The resulting fusion protein was designed by forming a site-to-site binding, that is, the C-terminal site of GST fused to the N-terminal site of CaM. The fused gene encodes a single polypeptide of GST and CaM. The sequences of nucleotides and resulting amino acids in the junction region between GST and CaM are shown in Figure 1. The fusion protein deduced from the DNA sequence has a molecular mass of ca. 43 kDa. The cell extracts obtained from E. coli JM105 harboring pGEX-CaM were analyzed by a 10% SDS-PAGE. Figure 2 shows SDS-PAGE analysis of cell extracts of host JM105 (lane 4), transformed cells with pGEX-CaM (lane 3), and IPTG-induced transformed cells (lane 2). From the bands of marker proteins (lane 1), the main band in the lane 2 was ascribed to the fusion protein with a molecular mass of ca. 43 kDa. The band was not observed in lanes 3 and 4. The protein was produced in large amounts in soluble form from only the transformed cells induced with IPTG. Purification of the Fusion Protein. The fusion protein could be purified in one step through the use of glutathione-Sepharose beads. About 6.4 mg of the purified fusion protein was obtained from 400 mL of culture. The purified fusion protein was analyzed by a 10% native PAGE, in the presence and in the absence of Ca2+ (Figure 3). The fusion protein could be purified with the affinity chromatography as shown in one band by staining with Coomassie brilliant blue. The migration in the presence of Ca2+ (lane 1) was a little faster than that in the absence of Ca2+ (lane 2). This result indicates that the CaM moiety of the fusion protein binds Ca2+ and changes its conformation to a compact folding. In the fusion protein, there is a recognition site of factor Xa, which is a site specific protease, between GST and CaM. So the GST moiety and CaM moiety could be separated easily from the fusion protein by cutting off with factor Xa. Ca2+ Dependent PDE Activation by the Fusion Protein. PDE is one of the typical CaM dependent enzymes. This enzyme is activated in the presence of Ca2+-binding CaM and catalyzes the hydrolysis of cAMP to AMP. CaM-deficient PDE will catalyze this reaction at a low rate, and CaM activates PDE only in the presence of Ca2+. Thus, the rate of reaction of PDE can
Kobatake et al.
Figure 3. PAGE analysis of the purified fusion protein. The fusion protein was purified with glutathione-Sepharose beads: lane 1, the purified fusion proten in the presence of Ca2+ (treated with 2 mM CaCl2); and lane 2, the fusion protein in the absence of Ca2+ [treated with 3 mM 1,2-bis(2-aminoethoxy)ethaneN,N,N′,N′-tetraacetic acid (EGTA)].
Figure 4. PDE activation by GST-CaM fusion protein. The fusion protein and PDE were incubated at 30 °C for 10 min in the assay mixture, in the absence [(-)Ca2+/GST-CaM] and presence [(+)Ca2+/GST-CaM] of Ca2+. PDE activity with CaM which was digested from the fusion protein by factor Xa was also measured [(+)Ca2+/CaM].
be used to estimate the amount of CaM present in the reaction mixture. Figure 4 shows the PDE activity with the fusion protein in the absence and presence of Ca2+. The activity of PDE in the presence of Ca2+ is about 4-fold higher than the activity in the absence of Ca2+. Moreover, the PDE activity with CaM (not fusing with GST) which was separated from the fusion protein by factor Xa was almost the same as the activity by the fusion protein. These results indicate that the CaM moiety in the fusion protein functioned as an activator of PDE without losing its activity by fusing with GST. Reversible Reactivity of Calmodulin on a Matrix. In order to confirm the possibility for on-off switching of enzymatic activity by CaM with or without Ca2+, the fusion protein was immobilized on a solid-phase matrix. The glutathione-Sepharose beads were used as a matrix for immobilization of the fusion protein through the use of the GST moiety. Then the PDE activity which is modulated by immobilized CaM was investigated. A
On−Off Switching of Enzymatic Reaction
Bioconjugate Chem., Vol. 7, No. 1, 1996 129
toward a molecular assembly, the data obtained in this study will open a new field for assembly of functional protein. Such Ca2+ responsible molecular assembly may be useful as a bioreactor system or new intelligent materials for the delivery of drugs such as calcitonine and parathyloid hormone, which are responsible for the regulation of Ca2+ concentration in serum. LITERATURE CITED
Figure 5. PDE activation by GST-CaM fusion protein in immobilized form. The fusion protein on the solid-phase matrix was incubated with PDE assay mixture. The reaction mixture in the presence (+) and absence (-) of Ca2+ was incubated alternately, and PDE activity in each stage was determined.
solution of the glutathione beads (50 µL) which immobilized the fusion protein was used instead of a CaM solution in the assay mixture described in Experimental Procedures. After incubation for 10 min at 30 °C, cAMP was added to a final concentration of 87 mM. The change in absorbance for 10 min at 265 nm was measured, and PDE activity was determined. Figure 5 shows the PDE activity with the fusion protein in the presence and in the absence of Ca2+. In the presence of Ca2+, the PDE activity was almost the same as that activated by free CaM (comparing with Figure 3). Then the beads were washed three times with 100 mM glycylglycine buffer (pH 7.5) containing 3 mM EGTA for chelating Ca2+. After centrifugation, the beads were resuspended in 970 mL of the reaction buffer containing EGTA instead of CaCl2, and then a solution of cAMP was added. In this case, the PDE activity was restricted at a lower level. Then the beads were washed again with 100 mM glycylglycine buffer (pH 7.5) and suspended in a reaction buffer containing 2 mM CaCl2. The PDE activity was recovered to the initial level. These experiments were repeated three times. Almost the same PDE activity could be obtained in each stage of the experiment. These results suggest that the fusion protein could be used repeatedly by immobilization on a solid-phase matrix. SUMMARY
The present paper describes an approach toward construction of a system of controlling an enzymatic activity continuously and reversibly by assembling a modulating protein. For this purpose, GST-CaM fusion protein was selected and prepared by a genetic method. The resulting fusion protein had a binding affinity to matrix-bound glutathione with GST and a modulating activity with CaM in its immobilized form. One of the CaM dependent enzymes, PDE, was activated in a reversible manner by matrix-bound CaM with or without Ca2+. On-off switching of enzymatic reaction by Ca2+ was demonstrated. This also indicates the possibility of the repeated use of CaM for the CaM dependent enzymes. Although the work present here is a simple approach
(1) Bu¨low, L. (1987) Characterization of an artificial bifunctional enzyme, β-galactosidase/galactokinase, prepared by gene fusion. Eur. J. Biochem. 163, 443-448. (2) Ljungcrantz, P., Carlsson, H., Mansson, M.-O., Buckel, P., Mosbach, K., and Bu¨low, L. (1989) Construction of an Artificial Bifunctional Enzyme, β-Galactosidase/Galactose Dehydrogenase, Exhibiting Efficient Galactose Channeling. Biochemistry 28, 8786-8792. (3) Peterhans, A., Mecklenburg, M., Meussdoerffer, F., and Mosbach, K. (1987) A simple competitive enzyme-linked immunosorbent assay using antigen-β-galactosidase fusions. Anal. Biochem. 163, 470-475. (4) Lindbladh, C., Persson, M., Bu¨low, L., Stahl, S., and Mosbach, K. (1987) The design of a simple competitive ELISA using human proinsulin-alkaline phosphatase conjugate prepared by gene fusion. Biochem. Biophys. Res. Commun. 149, 607-614. (5) Kobatake, E., Nishimori, Y., Ikariyama, Y., Aizawa, M., and Kato, S. (1990) Application of a fusion protein, metapyrocatechase/protein A, to an enzyme immunoassay. Anal. Biochem. 186, 14-18. (6) Kobatake, E., Iwai, T., Ikariyama, Y., and Aizawa, M. (1993) Bioluminescent immunoassay with a protein A-luciferase fusion protein. Anal. Biochem. 208, 300-305. (7) Marston, A. O. (1986) The purification of eukariotic polypeptides synthesized in Escherichia coli. Biochem. J. 240, 1-12. (8) Maina, C. V., Riggs, P. D., Grandea, A. G., III, Slatko, B. E., Moran, L. S., Taigliamonte, J. A., McReynolds, L. A., and Guan, C. (1988) An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. Gene 74, 365-373. (9) Smith, D. B., and Johnson, K. S. (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67, 31-40. (10) Weetall, H. H., and Suzuki, S., Eds. (1975) Immobilized Enzyme Technology, Plenum Press, New York. (11) Babu, Y. S., Sack, J. S., Greenhough, T. J., Bugg, C. E., Means, A. R., and Cook, W. J. (1985) Three-dimensional structure of calmodulin. Nature 315, 37-40. (12) Yoshino, H., Minari, O., Matsushima, N., Ueki, T., Miyake, Y., Matsuo, T., and Izumi, Y. (1989) Calcium-induced shape change of calmodulin with mastoparan studies by solution X-ray scattering. J. Biol. Chem. 264, 19706-19709. (13) Kakiuchi, S., Hidaka, H., and Means, A. R. (1982) Calmodulin and Intracellular Ca2+ Receptors, Plenum Press, New York. (14) Klee, C. B., and Vanaman, T. C. (1982) Calmodulin. Adv. Protein Chem. 35, 213-321. (15) Watterson, D. M., and Vanaman, T. C. (1976) Affinity chromatography purification of a cyclic nucleotide phosphodiesterase using immobilized molecular protein, a troponin c-like protein from brain. Biochem. Biophys. Res. Commun. 73, 40-46. (16) Klee, C. B., and Krinks, M. H. (1978) Purification of Cyclic 3′,5′-Nucleotide Phosphodiesterase Inhibitory Protein by Affinity Chromatography on Activator Protein Coupled to Sepharose. Biochemistry 17, 120-126. (17) Kincaid, R. L., and Vaughan, M. (1983) Affinity Chromatography of Brain Nucleotide Phosphodiesterase Using 3-(2Pyridyldithio)propionyl-Substituted Calmodulin Linked to Thiol-Sepharose. Biochemistry 22, 826-830.
130 Bioconjugate Chem., Vol. 7, No. 1, 1996 (18) Damrongchai, N., Kobatake, E., Haruyama, T., Ikariyama, Y., and Aizawa, M. (1995) Calcium Responsive Two-Dimensional Molecular Assembling of Lipid-Conjugated Calmodulin. Bioconjugate Chem. 6, 264-268. (19) Kato, S., Sekine, S., Oh, S.-W., Kim, N.-S., Umezawa, Y., Abe, N., Yokoyama-Kobayashi, M., and Aoki, T. (1994) Construction of a human full-length cDNA bank. Gene 150, 243-250. (20) Nagai, K., and Thogersen, H. C. (1984) Generation of β-globin by sequence-specific proteolysis of a hybrid protein produced in Escherichia coli. Nature 309, 810-812.
Kobatake et al. (21) Kakiuchi, S., and Yamazaki, R. (1970) Calcium dependent phosphodiesterase activity and its activating factor (PAF) from brain: studies on cyclic 3′,5′-nucleotide phosphodiesterase (III). Biochem. Biophys. Res. Commun. 41, 1104-1110. (22) Schiefer, S. (1985) In Methods of Enzymatic Analysis, Vol. 9, pp 317-331, VCH, Deerfield Beach, FL, Weinheim, Germany.
BC9500857
