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May 1, 1996 - Applications of novel affinity cassette methods: use of peptide fusion handles for the purification of recombinant proteins. Milton T W ...
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Anal. Chem. 1996, 68, 1550-1555

Affinity Chromatography of Recombinant Peptides/ Proteins Based on a Calmodulin Fusion Tail Nathaniel G. Hentz, Vesna Vukasinovic, and Sylvia Daunert*

Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055

An affinity chromatography system has been developed for the separation of recombinant fusion proteins based on the Ca2+-dependent binding of calmodulin (CaM) to the drug phenothiazine. Specifically, in the presence of Ca2+, a recognition site for phenothiazine is exposed on calmodulin, allowing the binding of this drug to CaM. Upon removal of Ca2+ with EGTA, the conformation of calmodulin changes, and the phenothiazine-CaM complex dissociates. This Ca2+-dependent binding has been exploited in the development of a fusion tail approach for the affinity purification of recombinant proteins and peptides. Protein A (ProtA) was employed as a model protein to demonstrate the advantages of this approach. In particular, the developed affinity chromatography system was used to isolate several ProtA-CaM fusion proteins. These recombinant fusion proteins were expressed in Escherichia coli and Saccharomyces cerevisiae from appropriately designed plasmids. Four different plasmids (two each for the bacteria and yeast) were used that encoded the fusion of CaM to the immunoglobulin-binding portion of protein A. After expression of the fusion protein, the crude cell lysates were loaded onto the phenothiazine affinity column in the presence of a Ca2+-containing buffer. Upon elution with an EGTA buffer, the ProtA-CaM fusion protein was purified, as confirmed by SDS-PAGE electrophoresis and Western blot analysis. Affinity chromatography is a powerful purification method that can both reduce the number of separation steps and increase the purity of the isolated protein/peptide of interest.1,2 Affinity chromatography takes advantage of a strong, selective interaction between two members of an affinity pair (e.g., antigen-antibody, ligand-binding protein, inhibitor-enzyme, etc.). One of the components of the pair is usually immobilized on a chromatographic support and is used to purify its counterpart in a matrix that may contain numerous other compounds that do not interact with the affinity column. While this is a major advantage of this technique, conventional affinity chromatography is restricted to a limited number of proteins/peptides that can bind specifically to an affinity counterpart. Now, with recombinant DNA technology, a specific binding property can be introduced to the protein/ peptide of interest by genetically fusing an affinity tail to its N- or C-terminus.3 An affinity tail is designed to provide a unique binding property to the target protein/peptide that promotes (1) Wimalasena, R. L.; Wilson, G. S. LC-GC 1992, 10, 223-224. (2) Clonis, Y. D. Prog. Biotechnol. 1994, 9, 527-533. (3) Ford, C. F.; Souminen, I.; Glatz, C. E. Protein Expression Purif. 1991, 2, 95-107.

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interaction with an appropriately designed affinity column, leading to an efficient recovery with a high degree of purity. It is important to note that this technique is extremely valuable when the native protein/peptide of interest otherwise has no binding characteristics that would allow its efficient isolation in high purity. A variety of affinity tails that promote the efficient recovery of recombinant proteins have been proposed (for a review, see ref 3). Proteins with biotin attached on an affinity tail (directed by posttranslational modification) can take advantage of the (strept)avidin-biotin binding interaction.4 Also available as fusion tails are binding proteins that have an affinity for chromatographic columns containing immobilized polypeptides5-7 or carbohydrates.8,9 Other affinity tail systems include genetically fusing an enzyme or antigen to the protein of interest, which can then be purified on a column of immobilized substrate/inhibitor10,11 or monoclonal antibody,12-14 respectively. Finally, different types of genetically fused polyamino acid tails have been employed to purify proteins of interest by ion-exchange chromatography,15,16 immobilized metal affinity chromatography,17-19 hydrophobic interaction,20 or thiopropyl Sepharose20 columns. Thus, affinity chromatography can be used with proteins or peptides that have no specific binding properties in their native form. In the system described in this article, calmodulin was used as an affinity tail for the efficient purification of calmodulin conjugates prepared by chemical coupling and by gene fusion techniques. Calmodulin is a Ca2+-dependent binding protein with four Ca2+ binding sites that participates in a variety of regulatory processes. Calmodulin exhibits Ca2+-dependent binding toward a number of peptides21,22 and hydrophobic compounds,23 such as (4) Cronan, J. E. J. Biol. Chem. 1990, 265, 10327-10333. (5) Moks, T.; Abrahmse´n, L.; Holmgren, E.; Bilich, M.; Olsson, A.; Uhle´n, M.; Pohl, G.; Sterky, C.; Hultberg, H.; Josephson, S.; Holmgren, A.; Jo ¨rnvall, H.; Nilsson, B. Biochemistry 1987, 26, 5239-5244. (6) Stofko-Hahn, R.; Carr, D. W.; Scott, J. D. FEBS Lett. 1992, 302, 274-278. (7) Schmidt, T. G. M.; Skerra, A. J. Chromatogr. 1994, 676, 337-345. (8) Guan, C.; Li, P.; Riggs, P. D. Gene 1988, 67, 21-30. (9) Blondel, A.; Bedouelle, H. Eur. J. Biochem. 1990, 193, 325-330. (10) Ullmann, A. Gene 1984, 29, 27-31. (11) Guan, K.; Dixon, J. E. Anal. Biochem. 1991, 192, 262-267. (12) Field, J.; Nikawa, J. I.; Broek, D.; MacDonald, B.; Rodgers, L.; Wilson, I. A.; Lerner, R. A.; Wigler, M. Mol. Cell. Biol. 1988, 8, 2159-2165. (13) Hopp, T. P.; Prickett, K. S.; Price, V. L.; Libby, R. T.; March, C. J.; Cerretti, D. P.; Urdal, D. L.; Conlon, P. J. Bio/Technology 1988, 6, 1204-1210. (14) Knappik, A.; Pluckthun, A. BioTechniques 1994, 17, 754-761. (15) Brewer, S. J.; Sassenfeld, H. M. Trends Biotechnol. 1985, 3, 119-122. (16) Heng, M. H.; Glatz, C. E. J. Chromatogr. 1995, 689, 227-234. (17) Hochuli, E.; Bannwarth, W.; Do ¨beli, H.; Gentz, R.; Stu ¨ ber, D. Bio/Technology 1988, 6, 1321-1325. (18) Hutchens, T. W.; Yip, T. T. Anal. Biochem. 1990, 191, 160-168. (19) Todd, R. J.; Johnson, R. D.; Arnold, F. H. J. Chromatogr. 1994, 662, 1326. (20) Persson, M.; Bergstrand, M. G.; Bu ¨ low, L.; Mosbach, K. Anal. Biochem. 1988, 172, 330-337. (21) Tanaka, T.; Ohmura, T.; Hidaka, H. Mol. Pharmacol. 1982, 22, 403-407. 0003-2700/96/0368-1550$12.00/0

© 1996 American Chemical Society

the antipsychotic drug phenothiazine.24 In the presence of Ca2+, calmodulin changes conformation, allowing the binding to phenothiazine. Upon removal of Ca2+ with a complexing agent such as EDTA or EGTA, the conformation of the protein is altered in such a way that the phenothiazine recognition site is no longer available for binding, allowing the complete dissociation of the phenothiazine-calmodulin complex. The major advantage of this approach over several of the previously reported affinity tail techniques is the efficient purification of the targeted proteins under mild elution conditions, which significantly reduces the probability of denaturation of the protein. Moreover, unlike some other protein affinity tails, the calmodulin used in these studies contains no cysteine residues; this eliminates the possibility of disulfide bond formation between calmodulin and the target protein, which could complicate the purification process. EXPERIMENTAL SECTION Reagents. Ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA) and 2-(trifluoromethyl)phenothiazine were obtained from Aldrich (Milwaukee, WI). Ethylenediaminetetraacetic acid (EDTA) was purchased from Fisher Scientific (Fair Lawn, NJ). Adenosine 5′-triphosphate (ATP), cyanogen bromide (CNBr), dextran-FITC (Dex-F) (average MW 4300), calmodulin (CaM), calmodulin-FITC (CaM-F), kanamycin, phenylmethylsulfonyl fluoride, leupeptin, and molecular weight standards were obtained from Sigma (St. Louis, MO). 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and tris(hydroxymethyl)aminomethane (Tris) were purchased from Research Organics (Cleveland, OH). Tween 20 was obtained from Promega (Madison, WI). The horseradish peroxidase color development solution, containing 4-chloro-1-naphthol, was obtained from Bio-Rad (Melville, NY). Goat-raised antibody against human immunoglobulin G conjugated to horseradish peroxidase (IgGHRP) was purchased from Pierce (Rockford, IL). Luria-Bertani (LB) broth and YPD media (1% yeast extract, 2% peptone, and 2% dextrose) were obtained from Difco Laboratories (Detroit, MI). Deionized (Milli-Q water purification system, Millipore, Bedford, MA) distilled water was used to prepare all solutions and mobile phases. Preparation of Affinity Chromatography Support. 2-(Trifluoromethyl)-10H-(3′-aminopropyl)phenothiazine hydrochloride (TAPP) was synthesized from commercially available 2-(trifluoromethyl)phenothiazine according to the procedure of Hart et al.25 The synthesized TAPP was coupled to Protein-Pak affinity epoxy-activated silica packing material (40 µm diameter, 300 Å pore size) (Millipore, Milford, MA). The coupling reaction performed was essentially that of Charbonneau et al.26 The ratio of TAPP to dry epoxy-activated silica was 10 mg/g. The TAPPsilica affinity medium was stored in a solution of 10 mM TrisHCl, pH 7.5, containing 1 mM sodium azide, and kept in the dark at 4 °C. Conjugation of Dextran-FITC to Calmodulin. The procedure of cyanogen bromide activation described by Marshall and Rabinowitz27 was followed in the preparation of CaM-Dex-F conjugates using several ratios of Dex-F to CaM. The products (22) Maulet, Y.; Cox, J. A. Biochemistry 1983, 22, 5680-5686. (23) Levin, R. M.; Weiss, B. Mol. Pharmacol. 1977, 13, 690-697. (24) Foster, W. S; Jarrett, H. W. J. Chromatogr. 1987, 403, 99-107. (25) Hart, R. C.; Bates, M. D.; Cormier, M. J.; Rosen, G. M.; Conn, P. M. Methods Enzymol. 1983, 102, 195-199. (26) Charbonneau, H.; Hice, R; Hart, R. C.; Cormier, M. J. Methods Enzymol. 1983, 102, 17-39. (27) Marshall, J. J.; Rabinowitz, M. L. J. Biol. Chem. 1976, 251, 1081-1087.

were dialyzed against deionized water, lyophilized, dissolved in 1.0 mL of deionized water, and subsequently frozen at -20 °C until further use. Apparatus. The coupled silica-TAPP affinity medium was packed into an HR 5/10 glass column (Pharmacia Biotech, Uppsala, Sweden). The buffers were pumped with a Rainin Rabbit HP solvent delivery system (Woburn, MA) at a flow rate of 1.30 mL/min. A Rheodyne Model 7125 injector (Cotati, CA), equipped with a 20-µL injection loop, was used for all Dex-F conjugate sample injections. For the cellular lysates, a 150-µL injection loop was used. For the affinity chromatography of CaM-Dex-F and CaM-F, detection was carried out on a SPEX Fluorolog-2 spectrofluorometer (SPEX Industries, Edison, NJ) with a µ-fluorescence flow cell (20-µL cell volume; NSG Precision Cells, Farmingdale, NY). The excitation was set at 495 nm, while the fluorescence emission was monitored at 518 nm. In all other experiments, a Knauer Model 87 variable wavelength UV-visible absorbance detector set at 280 nm was used. The pumps and the absorbance detector were interfaced with a Macintosh Plus computer (Cupertino, CA) using Dynamax software (Rainin). For the expression of the protein A-calmodulin (ProtA-CaM) fusion proteins, a Marathon Model 21K/BR refrigerated bench top centrifuge (Fisher Scientific) and a Micro Speed Fuge SFR11K (Savant Instruments, Farmingdale, NY) were employed. A Sonic Dismembrator Model 50 (Fisher Scientific) was used to lyse all bacterial cells before isolating the ProtA-CaM fusion proteins. SDS-PAGE of the purified fusion protein fractions was carried out with a PhastSystem electrophoresis setup (Pharmacia Biotech, Uppsala, Sweden). To verify the presence of protein A in the purified fusion protein fractions, the PhastSystem was also used to perform a Western blot analysis. Preparation of Protein A-Calmodulin Fusion Proteins in Escherichia coli and Saccharomyces cerevisiae. E. coli strain K12X289 was prepared fresh and transformed using standard protocols28 with plasmids pALP1CaM or pDS100 (Figure 1) on LB plates containing 30 and 50 µg/mL of kanamycin. Transformed E. coli was incubated at 37 °C overnight in 40 mL of LB medium containing kanamycin. After centrifugation at 4000 rpm for 15 min, the pellets were washed in TS buffer (50 mM Tris-HCl, pH 7.6, containing 150 mM NaCl), centrifuged again, and dissolved in 3 mL of TS buffer. A volume of 30 µL of protease inhibitors (20 mM phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, and 20 mM EDTA) was added, and the cells were lysed with the sonic dismembrator for 5 min. Cellular debris was removed by centrifugation at 11 000 rpm for 10 min, and Tween 20 was added to the supernatant to give a final concentration of 0.05% (v/v) Tween 20. The plasmids pDTW1 and pALP2R (Figure 2) were used to express ProtA-CaM fusion proteins in the yeast cells. S. cerevisiae strain BJ 5459 was used for transformation of the two plasmids on YPD plates, and semen DNA was used as a carrier DNA. Transformed yeast was grown at 30 °C in 100 mL of YPD medium for 4 days until an absorbance of 0.5 was obtained at 600 nm. Cells were harvested by centrifugation at 5000 rpm for 5 min, washed with washing buffer (100 mM Tris-HCl, pH 8.5, containing 10 mM EDTA), resuspended in 1 mL of buffer per 100 mg of yeast sample (wet weight), and centrifuged for a second (28) Maniatis, T.; Fritsch, E. F.; Sambrook, J. Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989.

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Figure 1. Plasmids (A) pDS100 and (B) pALP1CaM used for expression of ProtA-CaM fusion proteins in E. coli.

Figure 2. Plasmids (A) pALP2R and (B) pDTW1 used for expression of ProtA-CaM fusion proteins in S. cerevisiae.

time at 5000 rpm for 5 min. The cells were then resuspended in 0.125 mL of TS buffer per 100 mg of sample (wet weight) and lysed by shaking with glass beads at 4 °C for 5 min. After the lysate was separated from the glass beads, the cellular debris was centrifuged at 11 000 rpm for 5 min, and the supernatant was collected. The supernatant was stored at -20 °C until further use. Purification of Recombinant Protein A-Calmodulin Fusion Proteins. After the bacterial and yeast cells were lysed, 0.15 mL of the crude lysate was injected onto the TAPP affinity column that had been preequilibrated with buffer A (10 mM HEPES-HCl, 0.5 mM CaCl2, pH 7.0). The column was then washed with buffer A, followed by elution with buffer B (10 mM HEPES-HCl, pH 7.0, containing 0.5 mM CaCl2 and 0.25 M NaCl) to remove nonspecifically adsorbed proteins. Finally, the ProtACaM fusion protein was eluted with buffer C (10 mM Tris-HCl, 5 mM EGTA, pH 8.0). Elution of proteins was monitored by UV absorbance at 280 nm. It should be noted that the only sample pretreatment employed was preinjection filtration, carried out by a 0.45 µm syringe filter (Alltech, Deerfield, IL). The purity of the recombinant ProtA-CaM fusion protein was verified by SDSPAGE on 12.5% polyacrylamide PhastGels (Pharmacia Biotech). The gels were developed by silver staining (Pharmacia Biotech). The presence of protein A in the SDS-PAGE bands was verified by a Western blot analysis using an IgG-HRP conjugate.

toward phenothiazine. Calmodulin belongs to a family of calciumbinding proteins that contain EF hand motifs. These EF hand motifs, located at the N- and C-domains of calmodulin, constitute the calcium binding sites. Upon binding of Ca2+, calmodulin undergoes a conformational change that distorts the central helix that connects the N- and C-domains, exposing a hydrophobic region in each domain.29,30 It is in these hydrophobic regions that phenothiazine binds with high affinity (Kd ≈ 10-6 M).23 This high affinity is also a result of an additional conformational change, where helices in each of the two domains of calmodulin partially shield phenothiazine from the solvent. When calcium is removed, the calmodulin-phenothiazine complex dissociates, and calmodulin assumes its original conformation. Stationary phases with immobilized phenothiazine have been used to purify calmodulin by taking advantage of this calcium-dependent binding.31 With recombinant DNA methods, namely gene fusion, this property of calmodulin can be employed to purify recombinant proteins. The use of calmodulin affinity tails for this purpose is described for the first time in this article. A phenothiazine affinity column was prepared by attaching TAPP to epoxy-activated silica. To determine whether calmodulin conjugated to other molecules would retain its binding ability toward the TAPP-silica affinity column, a conjugate of calmodulin

RESULTS AND DISCUSSION The affinity chromatography system described in this article takes advantage of the Ca2+-dependent binding of calmodulin 1552

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(29) Chattopadhyaya, R.; Meador, W. E.; Means, A. R.; Quiocho, F. A. J. Mol. Biol. 1992, 228, 1177-1192. (30) Cook, W. J.; Walter, L. J.; Walter, M. R. Biochemistry 1994, 33, 1525915265. (31) Jarrett, H. W. J. Chromatogr. 1986, 363, 456-461.

Figure 3. Chromatogram of a 20-µL injection of 1 mg/mL CaM-F. The Ca2+- containing buffer (buffer A) was used until 33 min (indicated by the arrow), after which the EGTA-containing buffer (buffer C) was used to elute the CaM-F. For chromatographic conditions, see text.

Figure 4. Chromatogram of a 20-µL injection of the 42:1 (CaMDexF) conjugate. Buffer C (with EGTA) replaced buffer A (with Ca2+) at 11.7 min (indicated by the arrow).

with FITC was used. This CaM-F conjugate was loaded with buffer A on the affinity column after the column was washed and equilibrated with the same buffer. The CaM-F conjugate was then eluted with buffer C. The fluorescence was monitored with an excitation wavelength of 495 nm and an emission wavelength of 518 nm. Free fluorescein was eluted after 1.7 min had elapsed. This was expected since free fluorescein has no Ca2+-dependent binding toward phenothiazine and, thus, is not retained by the column. The free fluorescein peak is not noticeable in the chromatogram due to its small fluorescence intensity relative to the CaM-F peak (Figure 3). CaM-F was eluted only with the EGTA-containing buffer (buffer C). The EGTA in buffer C strips Ca2+ from the CaM-F conjugate, causing the dissociation of the CaM-F complex with TAPP. This set of experiments demonstrates that the Ca2+-dependent binding of calmodulin is unaffected by the attachment of FITC. Once it had been demonstrated that conjugated calmodulin can bind to the TAPP column, a study was undertaken to determine whether the Ca2+-specific binding of calmodulin is affected when conjugated to larger molecular weight molecules. For this purpose, several conjugates between CaM and Dex-F (average MW ) 4300) were prepared. Specifically, mole ratios of 218:1, 42:1, 4.2:1, 2.1:1, and 0.8:1 Dex-F-CaM were employed in the conjugation reactions. Each of the five CaM-Dex-F conjugates was separated on the TAPP affinity column using conditions identical to those used for the CaM-F conjugate. Figure 4 shows the chromatogram of the 42:1 Dex-F-CaM conjugate. The peak at 3 min was observed during the washing of the column with Ca2+-containing buffer (buffer A) and can be attributed to unconjugated Dex-F.

Figure 5. Chromatogram of a 150-µL injection of a crude cellular lysate of E. coli, where plasmid pDS100 was expressed.

The data above indicate that conjugated calmodulin retains its recognition properties for the immobilized phenothiazine. It should, therefore, be possible to use calmodulin in the development of an affinity tail approach for the purification of recombinant proteins. This could be accomplished by preparing conjugates of calmodulin with the target protein, where the target protein is attached to the N-terminal amino acid of calmodulin by genetic means. The N-terminus of calmodulin was selected because it is away from all four of the Ca2+-binding sites as well as the phenothiazine binding site. Therefore, we hypothesized that a calmodulin affinity tail that is genetically fused to the target protein may not affect the properties of the protein (i.e., cause denaturation or permanent conformational change). To test this hypothesis, protein A was used as a model protein, and different recombinant ProtA-CaM fusion proteins were prepared. Four different plasmids were used for the expression of the ProtA-CaM fusion proteins: pALP1CaM (Figure 1A), pDS100 (Figure 1B), pALP2R (Figure 2A), and pDTW1 (Figure 2B). The plasmids have been constructed by fusion of the calmodulin gene CMD1 from S. cerevisiae to a portion of the spa gene of Staphylococcus aureus, which encodes for the immunoglobulinbinding portion of protein A.32 The amino-terminal part of calmodulin is attached to the immunoglobulin-binding portion of protein A, while the carboxylic end of calmodulin remains free. This enables calmodulin to bind to the phenothiazine in the presence of Ca2+. Plasmids pALP1CaM and pALP2R code for the same ProtACaM fusion protein, albeit in different host species. The same is true for pDS100 and pDTW1. The two bacterial fusion proteins differ in that the ProtA-CaM expressed in E. coli from pALP1CaM (or in S. cerevisiae from pALP2R) lacks the 12 N-terminal amino acids encoded by native CMD1, while the ProtA-CaM fusion protein expressed in E. coli from pDS100 (or pDTW1 when expressed in S. cerevisiae) contains all of the amino acids of native yeast calmodulin.32 In addition, the latter fusion protein also contains a flexible spacer peptide of 10 amino acids between the protein A and calmodulin portions of the fusion protein. After the recombinant fusion proteins were expressed in the appropriate host (E. coli or S. cerevisiae), the crude cell lysates were subjected to purification on the TAPP affinity column. Elution of the expressed ProtA-CaM fusion proteins was monitored by UV absorbance at 280 nm. Representative chromatograms of the ProtA-CaM expressed in E. coli and S. cerevisiae are shown in Figures 5 and 6. In all chromatograms, elution with buffer A, containing Ca2+, gave high absorbance due to the (32) Stirling, D. A.; Petrie, A.; Pulford, D. J.; Paterson, D. T. W.; Stark, M. J. R. Mol. Microbiol. 1992, 6, 703-713.

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Figure 6. Chromatogram of a 150-µL injection of a crude cellular lysate of S. cerevisiae, where plasmid pALP2R was expressed.

presence of cellular proteins and cell components, which do not have affinity for TAPP. As described by Charbonneau et al.,26 phenothiazine columns may also exhibit ion-exchange effects toward proteins. Therefore, to remove any impurities bound to the column through ionic interactions, buffer B was used. As the chromatograms in Figures 5 and 6 show, the presence of NaCl in buffer B released proteins not eluted with buffer A. Finally, the Ca2+-deficient buffer C (with EGTA) was used to elute the purified recombinant ProtA-CaM fusion protein from the TAPP column. The combination of buffers B and C also regenerates the column by releasing all adsorbed proteins. It should be noted that the calmodulin that lacked the 12 amino acids was separated as efficiently as the full-length one. In addition, these experiments also indicated that, at least for the purification of protein A, the addition of a spacer between ProtA and CaM is not necessary. Equally important is the finding that the incorporation of a flexible spacer does not interfere with the interaction of calmodulin and the TAPP affinity column. Thus, such a spacer peptide can be genetically inserted in calmodulin to allow the correct folding of the target protein, if necessary. Noting that E. coli contains thousands of intracellular proteins and approximately 100 periplasmic proteins33 and yeast an even larger number of proteins, the purity of each ProtA-CaM fraction was verified by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE). The full length ProtA-CaM (from pDS100) has a predicted molecular mass of 34 kDa; however, the protein band has a higher apparent mobility and appears at approximately 32 kDa. This phenomenon was also observed by Stirling et al.32 An additional band, observed at 27 kDa (lane 3, Figure 7), was attributed to a proteolysis product of the ProtACaM fusion protein. It has been shown previously that proteolysis of recombinant protein A or protein A fusion proteins does occur frequently in E. coli.34-37 The pALP1CaM fusion protein resulted in a band of slightly greater mobility (lane 4, Figure 7), which was expected since the pALP1CaM fusion protein lacked 12 N-terminal amino acids. The pALP1CaM fusion protein seemed to be less susceptible to proteolysis than the pDS100 immediately after the cells were lysed. At approximately 68 kDa (lane 2, Figure 7), a faint band exists that is most likely due to the heat shock protein DnaK (∼69 kDa). This band was a result of running the (33) Pugsley, P. P.; Schwartz, M. FEMS Microbiol. Rev. 1985, 32, 3-38. (34) Ohya, Y.; Uno, I.; Ishikawa, T.; Anraku, Y. Eur. J. Biochem. 1987, 168, 13-19. (35) Nilsson, B.; Abrahmse´n, L.; Uhle´n, M. EMBO J. 1985, 4, 1075-1080. (36) Hellebust, H.; Uhle´n, M.; Enfors, S.-O. J. Bacteriol. 1990, 172, 5030-5034. (37) Yang, S.; Bergman, T.; Veide, A.; Enfors, S.-O. Eur. J. Biochem. 1994, 226, 847-852.

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Figure 7. SDS-PAGE gel demonstrating the purity of the fusion proteins, where lane 5 contains the molecular weight markers and lanes 4 and 3 contain the pALP1CaM and the pDS100 fusion proteins, respectively, after elution with the ATP-containing buffer. The DnaK is shown as a contaminant (in lane 2, at ∼67 kDa) of the pDS100 fusion protein when no ATP is used. Lane 1 shows the crude cellular lysate containing the pDS100 CaM-ProtA fusion protein.

affinity chromatography step without any ATP present in the elution buffers. Recombinant protein A, when expressed in E. coli, has been shown to bind to DnaK,36,37 and under certain conditions the two proteins can be copurified as a complex. It has also been shown that the DnaK-ProtA complex can be dissociated by ATP, a property that was employed in our system to release DnaK before eluting the ProtA-CaM fusion protein. It should be noted that the two additional SDS-PAGE bands, corresponding to a decomposition product of ProtA-CaM and to DnaK, are unique to ProtA systems and have not been observed with other CaM fusion proteins prepared in our laboratory (unpublished data). Next, a Western blot analysis was carried out on the polyacrylamide gel to verify whether the protein at 32 kDa was indeed a fusion of protein A. By using an IgG-HRP conjugate that binds to protein A, it was confirmed that the protein band at 32 kDa did contain a protein A fusion. This Western blot analysis also confirmed that the second band, observed around 27 kDa, was a proteolysis product of ProtA-CaM, since it also had affinity for the IgG-HRP conjugate. By using genetic engineering, an affinity tail can be attached with great precision to a target protein. Furthermore, once the plasmid has been designed and inserted into the host organism, the fusion protein can be produced in vivo. The affinity tail system described in this article demonstrates the feasibility of using a genetically engineered calmodulin tail for the purification of recombinant proteins/peptides. In particular, this affinity chromatography system provided an efficient method for the purification of several recombinant ProtA-CaM fusion proteins under mild conditions. It should be noted that, although phenothiazine is susceptible to light degradation, the phenothiazine-based chromatographic medium was found to be stable for several months (at least 6 months under continuous use) when protected

from light. It should also be noted that the experiments were performed under the worse possible conditions, where crude cellular lysate was injected for chromatographic analysis. In addition to the purification of the fusion proteins, protein-protein interaction studies may be possible if the fusion protein is used as a bifunctional affinity ligand. This will allow for the identification of cellular components that can interact with the protein fused to calmodulin as well as in the screening of drugs that can inhibit or promote specific protein-protein interactions. This chromatographic method can be generalized to include other CaM-protein fusion systems that incorporate a selective endopeptidase cleavage site between the calmodulin and the protein of interest (currently underway in our laboratory). After the affinity chromatography step, the target protein can be isolated from the fusion protein through treatment with the appropriate endopeptidase. Note that it must be determined for each of the other target proteins to be attached to the calmodulin affinity tail

whether the biological activity of the calmodulin affinity tail is affected. ACKNOWLEDGMENT This work was supported in part by the National Aeronautics and Space Administration (NCCW-60) and the National Institutes of Health (GM 47915). The plasmids pALP1CaM, pDS100, pDTW1, and pALP2R were a gift of D. A. Stirling,32 and E. coli strain K12X289 (F-, strs, T6s, T3r, l-) was kindly provided by S. C. Straley. S. cerevisiae strain BJ 5459 (a, ura 3-52, trp 1, lys 2-801, leu 2∆1, his 3∆200, pep 4::His 3, prb 1∆1-6R, can 1, GAL) was obtained from B. Raymond. Received for review October 9, 1995. Accepted February 6, 1996.X AC951022K X

Abstract published in Advance ACS Abstracts, March 15, 1996.

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