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Bacterial Expression of Human Vitamin D-Binding Protein (Gc2) in Functional Form
PROTEIN EXPRESSION AND PURIFICATION ARTICLE NO.
10, 115–122 (1997)
PT960720
Bacterial Expression of Human Vitamin D-Binding Protein (Gc2) in Functional Form1,2 Narasimha Swamy,* Sujoy Ghosh,† and Rahul Ray*,3 *Department of Bioorganic and Protein Chemistry, Vitamin D Laboratory, and Department of Physiology, Boston University School of Medicine, Boston, Massachusetts; and †Duke University Medical Center, Durham, North Carolina
Received October 29, 1996, and in revised form December 16, 1996
In this report, we report the first expression of human vitamin D-binding protein (hDBP), a serum protein with several functions and a multidomained structure, in Escherichia coli. The recombinant protein (reDBP) was expressed as a fusion partner of glutathione S-transferase in order to facilitate proper folding of the reDBP; E. coli-expressed DBP was found to be fully functional with respect to vitamin D sterol binding, interaction with actin, and cross-reactivity with anti-DBP antibody. Furthermore, both natural DBP and reDBP were affinity-labeled with 25-hydroxyvitamin D3-3-bromo[1-14C]acetate in a similar fashion. Availability of an expression system for hDBP in functional form provides opportunity to develop mutants and truncated DBPs to study multiple ligand-binding properties of this protein in relationship with its structure. q 1997 Academic Press
Vitamin D-binding protein (DBP) or Group Specific Component (Gc) is a genetically polymorphic and sparsely glycosylated (0 – 5%) serum protein (1 – 3). The two major phenotypes of Gc (DBP) are Gc1 and Gc2, which differ from each other in four amino acids (152, 311, 416, and 420) as well as by attached polysaccharide structure (4,5). The primary function of DBP involves highly specific binding (Kd Å 1008 – 11 M) and tissue-specific delivery of vitamin D and its metabolites. DBP binds vitamin D3 in serum, and transports this secosteroid to liver for the biosynthe1 Preliminary reports of this work were presented at the 9th Symposium of The Protein Society, July 8–12, 1995, Boston, MA, and the 17th Annual Meeting of the American Society for Bone and Mineral Research, September 9–13, 1995, Baltimore, MD. 2 This work is dedicated to the memory of Dr. Barbara H. Bowman. 3 To whom correspondence should be addressed at Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118. Fax: (617) 638-8882. E-mail: [email protected].
sis of 25-hydroxyvitamin D3 (25-OH-D3); the latter in turn is transported by DBP to kidney to form 1a,25dihydroxyvitamin D3 (1a,25(OH)2D3). 1a,25(OH)2D3 , the active form of vitamin D hormone, is finally translocated (also by DBP) to its primary target tissues, i.e., intestine, where it is responsible for intestinal calcium absorption, and to bone, where it stimulates bone calcium mobilization (1,2). DBP has several functions other than transportation of vitamin D and its metabolites. In conjunction with plasma gelsolin, it is involved in high-affinity sequestration of actin monomers, preventing clogging of arteries during cell injury and death (6,7). DBP also binds fatty acids with high affinity (8–10). Additionally, it enhances complement activation on neutrophil chemotaxis by binding to complement C5a and C5a des Arg (11). DBP is also known to be associated with B- and T-lymphocytes (12–14). Recently, it has been shown that DBP, after the in vivo processing of the glycan portion, acts as a potent macrophage and osteoclast activating factor (15,16). The primary structure of DBP has been determined by cDNA and amino acid sequencing. Comparison of these sequences has demonstrated that DBP is highly homologous with albumin (ALB), a-fetoprotein (AFP), and afamin (17 – 19). DBP contains a large number of cysteine (Cys) residues distributed evenly in the molecule, which are present in the form of disulfide bonds, providing a rigid structure. DBP, in close resemblance with ALB, is considered to have a cassette-like structure with three independent structural domains (Fig. 1). It has been postulated that different functions of DBP are elicited by different parts of the 458-aminoacid-long mature molecule. For instance, Haddad et al. (20) have employed actin-affinity chromatography of a tryptic digest of DBP to demonstrate that G-actin binding takes place via the C-terminus of DBP. This region of the protein has also been implicated in the binding of 115
1046-5928/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Amino acid sequence of human DBP in the format originally proposed by Brown for albumin (42).
fatty acids (9). On the other hand, affinity/photoaffinity labeling studies by our laboratory and others have strongly suggested that the vitamin D sterol-binding region of DBP is located exclusively in the N-terminus of the protein (20–26). Furthermore, selective chemical modifications of DBP have been carried out to identify Trp-145, also in the N-terminal region, as an essential amino acid for vitamin D sterol binding (27). The present study was undertaken to develop an expression system for full-length human DBP in its functional form.
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MATERIALS AND METHODS
The cDNA for hDBP (Gc2 isomorph) (17), a kind gift from the late Dr. Barbara Bowman and Dr. Funmei Yang (The University of Texas Health Sciences Center at San Antonio), was cloned at PstI site in pEMBL 18 for the purpose of general manipulation. This construct was designated as pEMBL–DBP. DNA restriction and modification enzymes were purchased from New England Biolabs (Beverley, MA). Polymerase chain reactions were performed using the PCR kit purchased
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from Gibco BRL (Gaithersburg, MD) according to the manufacturer’s specifications. Oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer (Model ABI 394, Foster City, CA). Plasmid DNA sequencing was performed using Applied Biosystems ABI dye-terminator sequencing kit designed for use on Applied Biosystems automated sequencer (ABI 373A). The amino acid sequence of reDBP was confirmed by N-terminal sequencing using an Applied Biosystems Peptide Sequencer. Peptide and DNA sequencing and DNA synthesis were carried out at the core peptide/ DNA sequencing facility, Center for Advanced Biomedical Research, Boston University School of Medicine. Antibody was raised against a synthetic peptide whose sequence was derived from human vitamin Dbinding protein (FNAKGPLLKKELSSFI, 369–384) (a kind gift from Dr. Michael Holick, Boston University School of Medicine, Boston, MA). Alkaline phosphatase-conjugated goat anti-rabbit IgG was purchased from Sigma Chemical Company (St. Louis, MO). 5Bromo-4-chloro-3*-indylphosphate p-toluidine (BCIP), nitro blue tetrazolium chloride (NBT), and Immobilon P were from Pierce Chemical Co. (Rockford, IL). Actin, glutathione, oxidized glutathione, and glutathione agarose were from Sigma Chemical Co., pGEX-4T-2 and pET-15B were purchased from Pharmacia Biotech, Inc. (Piscataway, NJ) and Novagen, Inc. (Madison, WI), respectively. pEMBL 18 was from Boehringer Mannheim (Indianapolis, IN). 25-Hydroxy vitamin D3 was a kind gift from Drs. Richard Gray and James Yager (Amoco Research Co., Naperville, IL). Isopropylthiogalactoside (IPTG) was from American Bioanalyticals (Natick, MA). All other chemicals used were purest available. 25-Hydroxy[26(27)-3H]vitamin D3 (sp act 28 Ci/mmol) and bromo[1-14C]acetic acid (sp act 18 mCi/ mmol) were obtained from Amersham Corp. (Arlington Heights, IL) and Sigma Chemical Co., respectively. 25-Hydroxyvitamin D3-3b-bromo[1-14C]acetate (14C25-OH-D3-3-bromoacetate, sp act 18 mCi/mmol) was synthesized by a modification of the published procedure (20). 25-OH-D3 was coupled with a molar equivalent of bromo[1-14C]acetic acid in the presence of an excess of dicyclohexylcarbodiimide and a catalytic amount of 4,4*-N-dimethylaminopyridine in anhydrous dichloromethane. The desired product was purified by preparative TLC on silica using 25% ethyl acetate in hexane as eluant. Native DBP was obtained from pooled human serum (American Red Cross, Dedham, MA) by affinity chromatography on a Sepharose matrix which is modified with a derivative of 25-OH-D3 (28). Construction of expression vectors pET–DBP and pGEX–DBP. Commercially available pET-15b and pGEX-4T-2 served as the starting plasmids for construction of expression vectors pET–DBP and pGEX–
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DBP, respectively. The pET-15b plasmid contains a strong and inducible T7 phage promotor and an efficient ribosome binding site. This vector also offers a 19-amino-acid-long N-terminal peptide tag with a stretch of 6 His and a thrombin-cleavage site to facilitate easy purification. The E. coli host BL21DE3 provided T7 RNA polymerase under lacUV5 control, inducible by IPTG. The pGEX-4T-2 plasmid contains a strong and inducible tac promotor which directs the expression of glutathione S-transferase (GST). DBP is expressed as a fusion partner of GST. This expression vector also contains the lac I gene for the purpose of increased stringency in control of expression. The E. coli K-12 strain TB1 served as host. Polymerase chain reaction (PCR) with pEMBL–DBP as template was used to generate a 1375-base-pair-long insert representing the coding sequence for full-length and mature hDBP (Leu1 –Leu458 without the signal peptide). The oligonucleotide primer-1 (5*-TGCTACTCGAGApchGAGAGGCCGGGATTATG-AAAAG-3*), corresponding to the 5* end of the sense strand, and oligonucleotide primer-2 (5*-GTTGGATCCTCAGGACTACAGGATATTCTT-3*), corresponding to the antisense strand at the 3* end of the coding sequence, served as primers in the PCR. The oligonucleotide primers aided in the incorporation of restriction sites XhoI at the 5* end and BamHI at the 3* end of the insert, and extraction of the coding sequence of the mature protein. The PCR product was digested with XhoI and BamHI, and cloned into the XhoI/BamHI site of pET-15b. This construct was designated as pET– DBP. The DNA manipulations were carried out using E. coli BL21 as host. The construct was transformed into BL21DE3 for the purpose of expression. The construction procedure for pGEX – DBP was similar to that for pET – DBP. The oligonucleotide primer-3 (5*-TGCTAGGATCCAGAGGCCGGG – ATTATGAAAAG-3 *) corresponding to the 5* end of the sense strand and primer 4 (5*-GTTCTCGAGTCAGGA-CTACAGGATATT-CTT-3 *) corresponding to the antisense strand at the 3 * end of the coding sequence served as primers in PCR using pEMBL – DBP to generate the insert representing the coding sequence for full-length and mature hDBP. The oligonucleotide primers aided in the incorporation of restriction sites BamHI at the 5* end and XhoI at the 3 * end of the insert. The PCR product was digested with BamHI and XhoI, and cloned into the corresponding sites in the pGEX-4T-2 expression vector. The construct was designated as pGEX – DBP (Fig. 2). Escherichia coli TB1 served as the host for both DNA manipulation and expression techniques. Bacterial growth conditions and hDBP expression. BL21DE3 cells carrying pET–DBP and TB1 cells carrying pGEX–DBP were grown to stationary phase at
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FIG. 2. Construction of pGEX–DBP expression vector.
377C in Luria broth (LB) containing 100 mg/ml ampicillin (Amp). The cultures were diluted 100 times with fresh LB–Amp and continued to grow until absorbance at 600 nm (A600) reached 0.4–0.6. The expression of fusion protein was induced by the addition of 1.0 mM final concentration of IPTG for pET–DBP and 0.5 mM IPTG for pGEX–DBP for 2 h to overnight. The culture temperature and durations were varied in different experiments based on the need. The protein expression was followed by SDS–PAGE of sample cells before and after induction. Preliminary experiments were conducted at 377C, and a considerable degradation of the
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fusion protein was observed upon purification. In order to minimize the degradation, the culture expansion as well as induction were carried out at 257C. Proper folding of the fusion protein was spontaneous. Purification of GST–DBP fusion protein and recombinant DBP (reDBP). The purification of the GST– DBP fusion protein was performed according to the manufacturer’s specifications, with required modifications. The cells were lysed in TBS (50 mM Tris-HCl buffer, pH 8.3, 150 mM NaCl), containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.2 mg/ml lysozyme, and 0.1% Triton X-100. The cells were disrupted by
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sonication and insoluble material was pelleted by centrifugation at 12,000g for 30 min at 47C. The clear cell lysate containing fusion protein was passed through a glutathione–agarose affinity column (Sigma), preequilibrated with TBS. The column was washed extensively with TBS to remove all unbound proteins, and bound GST–DBP was eluted with 20 mM oxidized glutathione in 100 mM Tris buffer, pH 8.3. The fusion protein was cleaved with thrombin (according to manufacturer’s specifications) and dialyzed against TBS to remove oxidized glutathione. Thrombin was removed by benzamidine–Sepharose chromatography (according to manufacturer’s specifications, Sigma). GST was separated from recombinant DBP (reDBP) using glutathione– Sepharose-4B affinity column. Sequence fidelities of the constructs (pET–DBP and pGEX–DBP) and reDBP were confirmed by DNA sequencing and N-terminal peptide sequencing. The protein concentrations were determined using Bradford Protein Assay (Bio-Rad, Inc., Richmond, CA). For the purpose of immunoblotting, proteins were transferred electrophoretically to an Immobilon P membrane (Pierce Chemical) using a Bio-Rad miniblotting apparatus according to the manufacturer’s instructions. After transfer, the membranes were treated according to a published procedure (29). The membranes were soaked in 1% BSA in TNT buffer (25 mM Tris-HCl, pH 7.5, 0.5 M NaCl) for 45 min at room temperature to block the excess sites. The blot was then incubated with 1:10,000 diluted primary antibody in 1% BSA in TNT for 1 h at room temperature. After washing with TNT buffer, the blot was incubated with 1:30,000 diluted secondary antibody (alkaline phosphatase-conjugated goat anti-rabbit IgG, Sigma Chemical Company) in 1% BSA in TNT for 1 h at room temperature. The blots were thoroughly washed with TNT buffer and bands were visualized using a BCIP–NBT substrate system (Pierce) as defined by the manufacturer. Binding assays. Competitive binding assays of reDBP and native DBP with 3H-25-OH-D3 were carried out according to a published procedure in which samples of reDBP and native DBP in Tris-HCl buffer, pH 8.3, were incubated with 3H-25-OH-D3 (2000 cpm, 0.07 pmol) and various doses of 25-OH-D3 (0.92–62 pmol) for 20 h at 47C followed by incubation with Dextrancoated charcoal (07C) for 15 min, centrifugation, and radioactive counting (25). Actin-binding assays. Actin-binding studies were carried in 5.0 mM Tris-HCl buffer, pH 8.3, containing 0.1 mM DTT, 0.1 mM ATP, and 0.1 mM CaCl2 at room temperature. Briefly, 5.0 mg of native or recombinant DBP was mixed with 5.0 mg of actin in the buffer at 257C for 30 min and analyzed on 7.5% native polyacrylamide gel electrophoresis (PAGE). The complex formation was followed by the shift in the mobility of DBP band (6,7).
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Affinity-labeling studies. Affinity labeling of reDBP and native DBP was carried out by incubating samples of proteins with 14C-25-OH-D3-3-bromoacetate (20,000 cpm, 0.7 nmol) in 50 mM Tris-HCl buffer, pH 8.3, in the presence and absence of 1.0 mg (2.5 nmol) of 25OH-D3 overnight at 47C followed by 4 h at 257C. The samples were analyzed on a 10% SDS gel. The radioactivity was followed by phosphorimaging. RESULTS AND DISCUSSION
Recent cloning and sequencing of the DBP gene (17,18,30) have shown that it spans about 42 kb, consisting of 13 exons that code for the complete preDBP with a signal peptide required for its secretion (31). The intron–exon distribution pattern of this gene, which is located on chromosome 4-long arm (q11–q22) in close proximity to ALB and AFP counterparts (32,33), is similar to those of ALB and AFP (31). It is well established that liver is the primary center for the expression of DBP (2,34), although other tissues like placenta, yolk sac, kidney, testis, and abdominal fat tissue are also shown to express this protein (2). The mechanism, involved in the regulation of DBP gene expression, is virtually unknown; however, it is believed to be different from those of ALB and AFP gene expressions based on acute-phase response studies (35). Although human serum ALB has been expressed in E. coli (36), Bacillus subtilis (37), and yeast (38), no attempt has been made so far to express the cDNA of hDBP. Obviously, DBP, with its 14 disulfide bonds and globular structure, poses a significant challenge for its expression, particularly in its functional form. Initially, we selected pET-15b as the expression vector, with its highly efficient T7 promotor, His tag for easy purification, and thrombin-cleavage site for efficient fusionprotein cleavage. Upon induction, there was a good level of reDBP, as determined by SDS–PAGE (results not shown). However, the reDBP was completely insoluble; all efforts to solubilize and refold the protein met with little success. We then decided to express the protein as a fusion partner of GST. Functional GST can be expressed independently in bacteria and is known to help proper folding of several other protein molecules when expressed as a fusion partner (39). Furthermore, the commercially available expression vector pGEX-4T-2 provides a GST expression system under the control of chemically inducible tac promotor (Fig. 2). The expression vector also provided a built-in thrombin-cleavage site positioned before the beginning of the foreign protein in order to conveniently separate the protein of interest from GST. However, positioning of the thrombin-cleavage site introduced a Gly-Ser sequence at the N-terminal of the protein of interest. Upon induction, the GST– DBP fusion protein was expressed, as judged by SDS– PAGE.
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FIG. 3. Analysis of reDBP. (A) SDS–PAGE of GST–DBP fusion protein (15 mg) after elution from glutathione–agarose affinity column (left lane). MW, molecular weight marker standards. (B) SDS–PAGE of reDBP, after cleavage from fusion protein and purification by affinity chromatography. Lane 1, Native hDBP (5 mg); lane 2, reDBP (5 mg). (C) Western blot analysis of native DBP (lane 1), and reDBP (lane 2).
The GST–DBP fusion protein was readily purified by glutathione–agarose affinity chromatography of the cell lysate of IPTG-induced bacteria, in a single step. While the fusion protein remained bound to the column, contaminating proteins were washed off effectively. The fusion protein was eluted using 20 mM oxidized glutathione. SDS–PAGE of the fusion protein revealed an Ç80-kDa band corresponding to the molecular weight of DBP and GST together, along with a small amount of a 31-kD band (Fig. 3A). This small band probably represents a degradation product of the fusion protein by cell proteases. Thrombin-cleavage of the purified fusion protein resulted in separation of GST from DBP. Thrombin and GST were separated by benzamidine–Sepharose chromatography and glutathione–Sepharose chromatography, respectively. The SDS–PAGE of the protein sample after cleavage and separation of GST revealed a single protein band of 52 kDa which comigrated with natural hDBP isolated from human blood (Fig. 3B, lanes 1 and 2 for natural and recombinant DBP, respectively). Immunoblotting was performed to assess the antigenic property of the reDBP. As shown in Fig. 3C, the reDBP preparation cross-reacted with anti-DBP antibody very similarly to the native protein isolated from human blood (Fig. 3C, lanes 1 and 2 for natural and recombinant DBP, respectively). Functional characterization of reDBP was performed by determining its ability to specifically bind 25-OHD3 , and comparing this ability with that of native DBP. Competitive binding assays of reDBP with a fixed amount of 3H-25-OH-D3 and various amounts of 25OH-D3 indicated that 3H-25-OH-D3 was displaced by 25-OH-D3 in a dose-dependent manner (Fig. 4). Almost identical results were obtained with native DBP, dem-
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onstrating that reDBP is very similar to its natural counterpart in terms of vitamin D sterol-binding. Another functional test of reDBP was carried out by studying its interaction with G-actin. As shown in Fig. 5, both reDBP and natural DBP formed a binary complex with G-actin as detected by native PAGE. Under the same experimental conditions native DBP was completely converted to DBP–actin complex (Fig. 5, lane 5), but complexation was 60–70% complete with reDBP (Fig. 5, lane 4). This difference in reactivity may be a reflection of a subtle structural difference between na-
FIG. 4. Competitive binding assays of native DBP and reDBP. Samples of either native DBP or reDBP were incubated with 3H-25OH-D3 (2000 cpm, 0.07 pmol) and various amounts (0.92–62 pmol) of 25-OH-D3 at 47C followed by incubation with Dextran-coated charcoal (07C) for 15 min, centrifugation, and radioactive counting.
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tive and recombinant forms. Further research is needed to investigate this phenomenon. Further functional characterization of reDBP was carried out by subjecting it to affinity labeling with 14C25-OH-D3-3-bromoacetate, an affinity analog of 25-OHD3 (20,40). It was observed that reDBP and native DBP were labeled covalently by 14C-25-OH-D3-3-bromoacetate with equal efficiency (Fig. 6, lanes 1 and 2, respectively). This labeling was reduced drastically when the incubations were carried out in the presence of an excess of 25-OH-D3 (Fig. 6, lanes 3 and 4 for reDBP and native DBP, respectively). Radioactivity at the bottom of the gel represented the unreacted radiolabel. These results strongly suggested that the 25-OH-D3-binding pockets in reDBP and natural DBP were covalently modified by 14C-25-OH-D3-3-bromoacetate in a very similar fashion. The multifunctional nature of DBP, particularly in relation to its three-dimensional structure, is poorly understood. It has been speculated that the different ligand binding activities are separated spatially within the multidomained structure of DBP. The expression system described here provides an opportunity to express the full-length recombinant protein in its fully functional form, as well as its mutants. The same expression system can also be employed to obtain various parts and domains of the protein, which will be useful in various structure–function studies. Furthermore, the method described in this communication, produced an unglycosylated recombinant protein, the Gc2 isomorph of DBP. It was recently reported that the Gc2 isomorph, lacking any carbohydrate, produced significantly better quality crystals for X-ray diffraction studies than the glycosylated Gc1 variety (41). Hence, this method provides an easier and possibly better procedure for obtaining purified DBP which could potentially be used for determining the three-dimensional structures of the apo- and holo-protein.
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FIG. 6. Affinity labeling of reDBP and native hDBP by 14C-25-OHD3-3-bromoacetate: samples of reDBP and native DBP were incubated with 14C-25-OH-D3-3-bromoacetate in the presence or absence of an excess of 25-OH-D3 , followed by analysis on a 10% SDS gel. The radioactivity was analyzed by a phosphorimager. Lane 1, native DBP / 14C-25-OH-D3-3-bromoacetate; lane 2, reDBP / 14C-25-OHD3-3-bromoacetate; lane 3, native DBP / 14C-25-OH-D3-3-bromoacetate / 25-OH-D3; lane 4, reDBP / 14C-25-OH-D3-3-bromoacetate / 25-OH-D3 . Positions of standard molecular weight marker proteins are shown on the right.
ACKNOWLEDGMENTS The authors thank Mr. Ned Rich (Core peptide/DNA sequencing facility, Center for Advanced Biomedical Research, Boston University School of Medicine) for his assistance in protein and DNA sequencing. Parts of this work were supported by grants (DK 44337 and 47418) from the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health.
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FIG. 5. Native PAGE analysis of G actin-binding by native and reDBP: samples of reDBP and native hDBP were incubated with actin and analyzed on a 7.5% native gel. Lane 5, native DBP–actin; lane 4, reDBP–actin. Native DBP (lane 3), reDBP (lane 2), and muscle actin (lane 1) were used as controls.
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agent for human vitamin D-binding protein. Arch. Biochem. Biophys. 319, 504–507. Roy, R., and Ray, R. (1995) Aminopropylation vitamin D hormone (1a,25-dihydroxyvitamin D3), its biological precursors, and other steroidal alcohols: An anchoring moiety for affinity studies of sterols. Steroids 60, 530–533. Swamy, N., Brisson, M., and Ray, R. (1995) Trp-145 is essential for the binding of 25-hydroxyvitamin D3 to human serum vitamin D-binding protein. J. Biol. Chem. 270, 2636–2639. Swamy, N., Roy, A., Chang, R., Brisson, M., and Ray, R. (1995) Affinity purification of human plasma vitamin D-binding protein. Protein Express. Purif. 6, 185–188. Wong, S. W., Syvaoja, J., Tan, C. K., Downey, K. M., So, A. G., Linn, S., and Wang, T. S. (1989) DNA polymerases alpha and delta are immunologically and structurally distinct. J. Biol. Chem. 264, 5924–5928. Braun, A., Kofler, A., Morawietz, S., and Cleve, H. (1993) Sequence and organization of the human vitamin D-binding protein gene. Biochim. Biophys. Acta 1216, 385–394. Witke, W. F., Gibbs, P. E., Zielinski, R., Yang, F., and Bowman, B. H. (1993) Complete structure of the human Gc gene: Differences and similarities between members of the albumin gene family. Genomics 16, 751–754. Weitkamp, L. R., Rucknagel, D. L., and Gershowitz, H. (1966) Genetic linkage between structural loci for albumin and group specific component (Gc). Am. J. Human Genet. 18, 559–571. Harper, M. E., and Dugaiczyk, A. (1983) Linkage of the evolutionarily-related serum albumin and alpha-fetoprotein genes within q11-22 of human chromosome 4. Am. J. Human Genet. 35, 565–572. Belanger, L., Roy, S., and Allard, D. (1994) New albumin gene 3* adjacent to the alpha 1-fetoprotein locus. J. Biol. Chem. 269, 5481–5484. Guha, C., Osawa, M., Werner, P. A., Galbraith, R. M., and Paddock, G. V. (1995) Regulation of human Gc (vitamin D-binding) protein levels: Hormonal and cytokine control of gene expression in vitro. Hepatology 21, 1675–1680. Lawn, R. M., Adelman, J., Bock, S. C., Franke, A. E., Houck, C. M., Najarian, R. C., Seeburg, P. H., and Wion, K. L. (1981) The sequence of human serum albumin cDNA and its expression in E. coli. Nucleic Acids Res. 9, 6103–6114. Saunders, C. W., Schmidt, B. J., Mallonee, R. L., and Guyer, M. S. (1987) Secretion of human serum albumin from Bacillus subtilis. J. Bacteriol. 169, 2917–2925. Okabayashi, K., Nakagawa, Y., Hayasuke, N., Ohi, H., Miura, M., Ishida, Y., Shimizu, M., Murakami, K., Hirabayashi, K., Minamino, H., Horii, H., Masaki, A., Sumi, A., Ohmura, T., and Kawabe, H. (1991) Secretory expression of human serum albumin gene in the yeast. J. Biochem. 110, 103–110. 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. Swamy, N., and Ray, R. (1996) Affinity labeling of rat serum vitamin D-binding protein Arch. Biochem. Biophys. 333, 139– 144. Verboven, C. C., De Bondt, H. L., De Ranter, C., Bouillon, R., and Van Baelen, H. (1995) Crystallization and X-ray investigation of vitamin D-binding protein from human serum. Identification of the crystal content. J. Steroid Biochem. Mol. Biol. 54, 11–14. Brown, J. R. (1976) Structural origins of mammalian albumin. Fed. Proc. 35, 2141–2144.
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Bacterial Expression of Human Vitamin D-Binding Protein (Gc2) in Functional Form1,2 Narasimha Swamy,* Sujoy Ghosh,† and Rahul Ray*,3 *Department of Bioorganic and Protein Chemistry, Vitamin D Laboratory, and Department of Physiology, Boston University School of Medicine, Boston, Massachusetts; and †Duke University Medical Center, Durham, North Carolina
Received October 29, 1996, and in revised form December 16, 1996
In this report, we report the first expression of human vitamin D-binding protein (hDBP), a serum protein with several functions and a multidomained structure, in Escherichia coli. The recombinant protein (reDBP) was expressed as a fusion partner of glutathione S-transferase in order to facilitate proper folding of the reDBP; E. coli-expressed DBP was found to be fully functional with respect to vitamin D sterol binding, interaction with actin, and cross-reactivity with anti-DBP antibody. Furthermore, both natural DBP and reDBP were affinity-labeled with 25-hydroxyvitamin D3-3-bromo[1-14C]acetate in a similar fashion. Availability of an expression system for hDBP in functional form provides opportunity to develop mutants and truncated DBPs to study multiple ligand-binding properties of this protein in relationship with its structure. q 1997 Academic Press
Vitamin D-binding protein (DBP) or Group Specific Component (Gc) is a genetically polymorphic and sparsely glycosylated (0 – 5%) serum protein (1 – 3). The two major phenotypes of Gc (DBP) are Gc1 and Gc2, which differ from each other in four amino acids (152, 311, 416, and 420) as well as by attached polysaccharide structure (4,5). The primary function of DBP involves highly specific binding (Kd Å 1008 – 11 M) and tissue-specific delivery of vitamin D and its metabolites. DBP binds vitamin D3 in serum, and transports this secosteroid to liver for the biosynthe1 Preliminary reports of this work were presented at the 9th Symposium of The Protein Society, July 8–12, 1995, Boston, MA, and the 17th Annual Meeting of the American Society for Bone and Mineral Research, September 9–13, 1995, Baltimore, MD. 2 This work is dedicated to the memory of Dr. Barbara H. Bowman. 3 To whom correspondence should be addressed at Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118. Fax: (617) 638-8882. E-mail: [email protected].
sis of 25-hydroxyvitamin D3 (25-OH-D3); the latter in turn is transported by DBP to kidney to form 1a,25dihydroxyvitamin D3 (1a,25(OH)2D3). 1a,25(OH)2D3 , the active form of vitamin D hormone, is finally translocated (also by DBP) to its primary target tissues, i.e., intestine, where it is responsible for intestinal calcium absorption, and to bone, where it stimulates bone calcium mobilization (1,2). DBP has several functions other than transportation of vitamin D and its metabolites. In conjunction with plasma gelsolin, it is involved in high-affinity sequestration of actin monomers, preventing clogging of arteries during cell injury and death (6,7). DBP also binds fatty acids with high affinity (8–10). Additionally, it enhances complement activation on neutrophil chemotaxis by binding to complement C5a and C5a des Arg (11). DBP is also known to be associated with B- and T-lymphocytes (12–14). Recently, it has been shown that DBP, after the in vivo processing of the glycan portion, acts as a potent macrophage and osteoclast activating factor (15,16). The primary structure of DBP has been determined by cDNA and amino acid sequencing. Comparison of these sequences has demonstrated that DBP is highly homologous with albumin (ALB), a-fetoprotein (AFP), and afamin (17 – 19). DBP contains a large number of cysteine (Cys) residues distributed evenly in the molecule, which are present in the form of disulfide bonds, providing a rigid structure. DBP, in close resemblance with ALB, is considered to have a cassette-like structure with three independent structural domains (Fig. 1). It has been postulated that different functions of DBP are elicited by different parts of the 458-aminoacid-long mature molecule. For instance, Haddad et al. (20) have employed actin-affinity chromatography of a tryptic digest of DBP to demonstrate that G-actin binding takes place via the C-terminus of DBP. This region of the protein has also been implicated in the binding of 115
1046-5928/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Amino acid sequence of human DBP in the format originally proposed by Brown for albumin (42).
fatty acids (9). On the other hand, affinity/photoaffinity labeling studies by our laboratory and others have strongly suggested that the vitamin D sterol-binding region of DBP is located exclusively in the N-terminus of the protein (20–26). Furthermore, selective chemical modifications of DBP have been carried out to identify Trp-145, also in the N-terminal region, as an essential amino acid for vitamin D sterol binding (27). The present study was undertaken to develop an expression system for full-length human DBP in its functional form.
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MATERIALS AND METHODS
The cDNA for hDBP (Gc2 isomorph) (17), a kind gift from the late Dr. Barbara Bowman and Dr. Funmei Yang (The University of Texas Health Sciences Center at San Antonio), was cloned at PstI site in pEMBL 18 for the purpose of general manipulation. This construct was designated as pEMBL–DBP. DNA restriction and modification enzymes were purchased from New England Biolabs (Beverley, MA). Polymerase chain reactions were performed using the PCR kit purchased
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from Gibco BRL (Gaithersburg, MD) according to the manufacturer’s specifications. Oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer (Model ABI 394, Foster City, CA). Plasmid DNA sequencing was performed using Applied Biosystems ABI dye-terminator sequencing kit designed for use on Applied Biosystems automated sequencer (ABI 373A). The amino acid sequence of reDBP was confirmed by N-terminal sequencing using an Applied Biosystems Peptide Sequencer. Peptide and DNA sequencing and DNA synthesis were carried out at the core peptide/ DNA sequencing facility, Center for Advanced Biomedical Research, Boston University School of Medicine. Antibody was raised against a synthetic peptide whose sequence was derived from human vitamin Dbinding protein (FNAKGPLLKKELSSFI, 369–384) (a kind gift from Dr. Michael Holick, Boston University School of Medicine, Boston, MA). Alkaline phosphatase-conjugated goat anti-rabbit IgG was purchased from Sigma Chemical Company (St. Louis, MO). 5Bromo-4-chloro-3*-indylphosphate p-toluidine (BCIP), nitro blue tetrazolium chloride (NBT), and Immobilon P were from Pierce Chemical Co. (Rockford, IL). Actin, glutathione, oxidized glutathione, and glutathione agarose were from Sigma Chemical Co., pGEX-4T-2 and pET-15B were purchased from Pharmacia Biotech, Inc. (Piscataway, NJ) and Novagen, Inc. (Madison, WI), respectively. pEMBL 18 was from Boehringer Mannheim (Indianapolis, IN). 25-Hydroxy vitamin D3 was a kind gift from Drs. Richard Gray and James Yager (Amoco Research Co., Naperville, IL). Isopropylthiogalactoside (IPTG) was from American Bioanalyticals (Natick, MA). All other chemicals used were purest available. 25-Hydroxy[26(27)-3H]vitamin D3 (sp act 28 Ci/mmol) and bromo[1-14C]acetic acid (sp act 18 mCi/ mmol) were obtained from Amersham Corp. (Arlington Heights, IL) and Sigma Chemical Co., respectively. 25-Hydroxyvitamin D3-3b-bromo[1-14C]acetate (14C25-OH-D3-3-bromoacetate, sp act 18 mCi/mmol) was synthesized by a modification of the published procedure (20). 25-OH-D3 was coupled with a molar equivalent of bromo[1-14C]acetic acid in the presence of an excess of dicyclohexylcarbodiimide and a catalytic amount of 4,4*-N-dimethylaminopyridine in anhydrous dichloromethane. The desired product was purified by preparative TLC on silica using 25% ethyl acetate in hexane as eluant. Native DBP was obtained from pooled human serum (American Red Cross, Dedham, MA) by affinity chromatography on a Sepharose matrix which is modified with a derivative of 25-OH-D3 (28). Construction of expression vectors pET–DBP and pGEX–DBP. Commercially available pET-15b and pGEX-4T-2 served as the starting plasmids for construction of expression vectors pET–DBP and pGEX–
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DBP, respectively. The pET-15b plasmid contains a strong and inducible T7 phage promotor and an efficient ribosome binding site. This vector also offers a 19-amino-acid-long N-terminal peptide tag with a stretch of 6 His and a thrombin-cleavage site to facilitate easy purification. The E. coli host BL21DE3 provided T7 RNA polymerase under lacUV5 control, inducible by IPTG. The pGEX-4T-2 plasmid contains a strong and inducible tac promotor which directs the expression of glutathione S-transferase (GST). DBP is expressed as a fusion partner of GST. This expression vector also contains the lac I gene for the purpose of increased stringency in control of expression. The E. coli K-12 strain TB1 served as host. Polymerase chain reaction (PCR) with pEMBL–DBP as template was used to generate a 1375-base-pair-long insert representing the coding sequence for full-length and mature hDBP (Leu1 –Leu458 without the signal peptide). The oligonucleotide primer-1 (5*-TGCTACTCGAGApchGAGAGGCCGGGATTATG-AAAAG-3*), corresponding to the 5* end of the sense strand, and oligonucleotide primer-2 (5*-GTTGGATCCTCAGGACTACAGGATATTCTT-3*), corresponding to the antisense strand at the 3* end of the coding sequence, served as primers in the PCR. The oligonucleotide primers aided in the incorporation of restriction sites XhoI at the 5* end and BamHI at the 3* end of the insert, and extraction of the coding sequence of the mature protein. The PCR product was digested with XhoI and BamHI, and cloned into the XhoI/BamHI site of pET-15b. This construct was designated as pET– DBP. The DNA manipulations were carried out using E. coli BL21 as host. The construct was transformed into BL21DE3 for the purpose of expression. The construction procedure for pGEX – DBP was similar to that for pET – DBP. The oligonucleotide primer-3 (5*-TGCTAGGATCCAGAGGCCGGG – ATTATGAAAAG-3 *) corresponding to the 5* end of the sense strand and primer 4 (5*-GTTCTCGAGTCAGGA-CTACAGGATATT-CTT-3 *) corresponding to the antisense strand at the 3 * end of the coding sequence served as primers in PCR using pEMBL – DBP to generate the insert representing the coding sequence for full-length and mature hDBP. The oligonucleotide primers aided in the incorporation of restriction sites BamHI at the 5* end and XhoI at the 3 * end of the insert. The PCR product was digested with BamHI and XhoI, and cloned into the corresponding sites in the pGEX-4T-2 expression vector. The construct was designated as pGEX – DBP (Fig. 2). Escherichia coli TB1 served as the host for both DNA manipulation and expression techniques. Bacterial growth conditions and hDBP expression. BL21DE3 cells carrying pET–DBP and TB1 cells carrying pGEX–DBP were grown to stationary phase at
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FIG. 2. Construction of pGEX–DBP expression vector.
377C in Luria broth (LB) containing 100 mg/ml ampicillin (Amp). The cultures were diluted 100 times with fresh LB–Amp and continued to grow until absorbance at 600 nm (A600) reached 0.4–0.6. The expression of fusion protein was induced by the addition of 1.0 mM final concentration of IPTG for pET–DBP and 0.5 mM IPTG for pGEX–DBP for 2 h to overnight. The culture temperature and durations were varied in different experiments based on the need. The protein expression was followed by SDS–PAGE of sample cells before and after induction. Preliminary experiments were conducted at 377C, and a considerable degradation of the
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fusion protein was observed upon purification. In order to minimize the degradation, the culture expansion as well as induction were carried out at 257C. Proper folding of the fusion protein was spontaneous. Purification of GST–DBP fusion protein and recombinant DBP (reDBP). The purification of the GST– DBP fusion protein was performed according to the manufacturer’s specifications, with required modifications. The cells were lysed in TBS (50 mM Tris-HCl buffer, pH 8.3, 150 mM NaCl), containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.2 mg/ml lysozyme, and 0.1% Triton X-100. The cells were disrupted by
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sonication and insoluble material was pelleted by centrifugation at 12,000g for 30 min at 47C. The clear cell lysate containing fusion protein was passed through a glutathione–agarose affinity column (Sigma), preequilibrated with TBS. The column was washed extensively with TBS to remove all unbound proteins, and bound GST–DBP was eluted with 20 mM oxidized glutathione in 100 mM Tris buffer, pH 8.3. The fusion protein was cleaved with thrombin (according to manufacturer’s specifications) and dialyzed against TBS to remove oxidized glutathione. Thrombin was removed by benzamidine–Sepharose chromatography (according to manufacturer’s specifications, Sigma). GST was separated from recombinant DBP (reDBP) using glutathione– Sepharose-4B affinity column. Sequence fidelities of the constructs (pET–DBP and pGEX–DBP) and reDBP were confirmed by DNA sequencing and N-terminal peptide sequencing. The protein concentrations were determined using Bradford Protein Assay (Bio-Rad, Inc., Richmond, CA). For the purpose of immunoblotting, proteins were transferred electrophoretically to an Immobilon P membrane (Pierce Chemical) using a Bio-Rad miniblotting apparatus according to the manufacturer’s instructions. After transfer, the membranes were treated according to a published procedure (29). The membranes were soaked in 1% BSA in TNT buffer (25 mM Tris-HCl, pH 7.5, 0.5 M NaCl) for 45 min at room temperature to block the excess sites. The blot was then incubated with 1:10,000 diluted primary antibody in 1% BSA in TNT for 1 h at room temperature. After washing with TNT buffer, the blot was incubated with 1:30,000 diluted secondary antibody (alkaline phosphatase-conjugated goat anti-rabbit IgG, Sigma Chemical Company) in 1% BSA in TNT for 1 h at room temperature. The blots were thoroughly washed with TNT buffer and bands were visualized using a BCIP–NBT substrate system (Pierce) as defined by the manufacturer. Binding assays. Competitive binding assays of reDBP and native DBP with 3H-25-OH-D3 were carried out according to a published procedure in which samples of reDBP and native DBP in Tris-HCl buffer, pH 8.3, were incubated with 3H-25-OH-D3 (2000 cpm, 0.07 pmol) and various doses of 25-OH-D3 (0.92–62 pmol) for 20 h at 47C followed by incubation with Dextrancoated charcoal (07C) for 15 min, centrifugation, and radioactive counting (25). Actin-binding assays. Actin-binding studies were carried in 5.0 mM Tris-HCl buffer, pH 8.3, containing 0.1 mM DTT, 0.1 mM ATP, and 0.1 mM CaCl2 at room temperature. Briefly, 5.0 mg of native or recombinant DBP was mixed with 5.0 mg of actin in the buffer at 257C for 30 min and analyzed on 7.5% native polyacrylamide gel electrophoresis (PAGE). The complex formation was followed by the shift in the mobility of DBP band (6,7).
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Affinity-labeling studies. Affinity labeling of reDBP and native DBP was carried out by incubating samples of proteins with 14C-25-OH-D3-3-bromoacetate (20,000 cpm, 0.7 nmol) in 50 mM Tris-HCl buffer, pH 8.3, in the presence and absence of 1.0 mg (2.5 nmol) of 25OH-D3 overnight at 47C followed by 4 h at 257C. The samples were analyzed on a 10% SDS gel. The radioactivity was followed by phosphorimaging. RESULTS AND DISCUSSION
Recent cloning and sequencing of the DBP gene (17,18,30) have shown that it spans about 42 kb, consisting of 13 exons that code for the complete preDBP with a signal peptide required for its secretion (31). The intron–exon distribution pattern of this gene, which is located on chromosome 4-long arm (q11–q22) in close proximity to ALB and AFP counterparts (32,33), is similar to those of ALB and AFP (31). It is well established that liver is the primary center for the expression of DBP (2,34), although other tissues like placenta, yolk sac, kidney, testis, and abdominal fat tissue are also shown to express this protein (2). The mechanism, involved in the regulation of DBP gene expression, is virtually unknown; however, it is believed to be different from those of ALB and AFP gene expressions based on acute-phase response studies (35). Although human serum ALB has been expressed in E. coli (36), Bacillus subtilis (37), and yeast (38), no attempt has been made so far to express the cDNA of hDBP. Obviously, DBP, with its 14 disulfide bonds and globular structure, poses a significant challenge for its expression, particularly in its functional form. Initially, we selected pET-15b as the expression vector, with its highly efficient T7 promotor, His tag for easy purification, and thrombin-cleavage site for efficient fusionprotein cleavage. Upon induction, there was a good level of reDBP, as determined by SDS–PAGE (results not shown). However, the reDBP was completely insoluble; all efforts to solubilize and refold the protein met with little success. We then decided to express the protein as a fusion partner of GST. Functional GST can be expressed independently in bacteria and is known to help proper folding of several other protein molecules when expressed as a fusion partner (39). Furthermore, the commercially available expression vector pGEX-4T-2 provides a GST expression system under the control of chemically inducible tac promotor (Fig. 2). The expression vector also provided a built-in thrombin-cleavage site positioned before the beginning of the foreign protein in order to conveniently separate the protein of interest from GST. However, positioning of the thrombin-cleavage site introduced a Gly-Ser sequence at the N-terminal of the protein of interest. Upon induction, the GST– DBP fusion protein was expressed, as judged by SDS– PAGE.
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FIG. 3. Analysis of reDBP. (A) SDS–PAGE of GST–DBP fusion protein (15 mg) after elution from glutathione–agarose affinity column (left lane). MW, molecular weight marker standards. (B) SDS–PAGE of reDBP, after cleavage from fusion protein and purification by affinity chromatography. Lane 1, Native hDBP (5 mg); lane 2, reDBP (5 mg). (C) Western blot analysis of native DBP (lane 1), and reDBP (lane 2).
The GST–DBP fusion protein was readily purified by glutathione–agarose affinity chromatography of the cell lysate of IPTG-induced bacteria, in a single step. While the fusion protein remained bound to the column, contaminating proteins were washed off effectively. The fusion protein was eluted using 20 mM oxidized glutathione. SDS–PAGE of the fusion protein revealed an Ç80-kDa band corresponding to the molecular weight of DBP and GST together, along with a small amount of a 31-kD band (Fig. 3A). This small band probably represents a degradation product of the fusion protein by cell proteases. Thrombin-cleavage of the purified fusion protein resulted in separation of GST from DBP. Thrombin and GST were separated by benzamidine–Sepharose chromatography and glutathione–Sepharose chromatography, respectively. The SDS–PAGE of the protein sample after cleavage and separation of GST revealed a single protein band of 52 kDa which comigrated with natural hDBP isolated from human blood (Fig. 3B, lanes 1 and 2 for natural and recombinant DBP, respectively). Immunoblotting was performed to assess the antigenic property of the reDBP. As shown in Fig. 3C, the reDBP preparation cross-reacted with anti-DBP antibody very similarly to the native protein isolated from human blood (Fig. 3C, lanes 1 and 2 for natural and recombinant DBP, respectively). Functional characterization of reDBP was performed by determining its ability to specifically bind 25-OHD3 , and comparing this ability with that of native DBP. Competitive binding assays of reDBP with a fixed amount of 3H-25-OH-D3 and various amounts of 25OH-D3 indicated that 3H-25-OH-D3 was displaced by 25-OH-D3 in a dose-dependent manner (Fig. 4). Almost identical results were obtained with native DBP, dem-
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onstrating that reDBP is very similar to its natural counterpart in terms of vitamin D sterol-binding. Another functional test of reDBP was carried out by studying its interaction with G-actin. As shown in Fig. 5, both reDBP and natural DBP formed a binary complex with G-actin as detected by native PAGE. Under the same experimental conditions native DBP was completely converted to DBP–actin complex (Fig. 5, lane 5), but complexation was 60–70% complete with reDBP (Fig. 5, lane 4). This difference in reactivity may be a reflection of a subtle structural difference between na-
FIG. 4. Competitive binding assays of native DBP and reDBP. Samples of either native DBP or reDBP were incubated with 3H-25OH-D3 (2000 cpm, 0.07 pmol) and various amounts (0.92–62 pmol) of 25-OH-D3 at 47C followed by incubation with Dextran-coated charcoal (07C) for 15 min, centrifugation, and radioactive counting.
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tive and recombinant forms. Further research is needed to investigate this phenomenon. Further functional characterization of reDBP was carried out by subjecting it to affinity labeling with 14C25-OH-D3-3-bromoacetate, an affinity analog of 25-OHD3 (20,40). It was observed that reDBP and native DBP were labeled covalently by 14C-25-OH-D3-3-bromoacetate with equal efficiency (Fig. 6, lanes 1 and 2, respectively). This labeling was reduced drastically when the incubations were carried out in the presence of an excess of 25-OH-D3 (Fig. 6, lanes 3 and 4 for reDBP and native DBP, respectively). Radioactivity at the bottom of the gel represented the unreacted radiolabel. These results strongly suggested that the 25-OH-D3-binding pockets in reDBP and natural DBP were covalently modified by 14C-25-OH-D3-3-bromoacetate in a very similar fashion. The multifunctional nature of DBP, particularly in relation to its three-dimensional structure, is poorly understood. It has been speculated that the different ligand binding activities are separated spatially within the multidomained structure of DBP. The expression system described here provides an opportunity to express the full-length recombinant protein in its fully functional form, as well as its mutants. The same expression system can also be employed to obtain various parts and domains of the protein, which will be useful in various structure–function studies. Furthermore, the method described in this communication, produced an unglycosylated recombinant protein, the Gc2 isomorph of DBP. It was recently reported that the Gc2 isomorph, lacking any carbohydrate, produced significantly better quality crystals for X-ray diffraction studies than the glycosylated Gc1 variety (41). Hence, this method provides an easier and possibly better procedure for obtaining purified DBP which could potentially be used for determining the three-dimensional structures of the apo- and holo-protein.
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FIG. 6. Affinity labeling of reDBP and native hDBP by 14C-25-OHD3-3-bromoacetate: samples of reDBP and native DBP were incubated with 14C-25-OH-D3-3-bromoacetate in the presence or absence of an excess of 25-OH-D3 , followed by analysis on a 10% SDS gel. The radioactivity was analyzed by a phosphorimager. Lane 1, native DBP / 14C-25-OH-D3-3-bromoacetate; lane 2, reDBP / 14C-25-OHD3-3-bromoacetate; lane 3, native DBP / 14C-25-OH-D3-3-bromoacetate / 25-OH-D3; lane 4, reDBP / 14C-25-OH-D3-3-bromoacetate / 25-OH-D3 . Positions of standard molecular weight marker proteins are shown on the right.
ACKNOWLEDGMENTS The authors thank Mr. Ned Rich (Core peptide/DNA sequencing facility, Center for Advanced Biomedical Research, Boston University School of Medicine) for his assistance in protein and DNA sequencing. Parts of this work were supported by grants (DK 44337 and 47418) from the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health.
REFERENCES
FIG. 5. Native PAGE analysis of G actin-binding by native and reDBP: samples of reDBP and native hDBP were incubated with actin and analyzed on a 7.5% native gel. Lane 5, native DBP–actin; lane 4, reDBP–actin. Native DBP (lane 3), reDBP (lane 2), and muscle actin (lane 1) were used as controls.
1. Cooke, N. E., and Haddad, J. G. (1995) Vitamin D-bindingprotein (Gc-globulin): Update. Endocr. Rev. 4, 125–128. 2. Cooke, N. E., and Haddad, J. G. (1989) Vitamin D binding protein (Gc-globulin). Endocr. Rev. 10, 294–307. 3. Ray, R. (1996) Molecular recognition in vitamin D-binding protein. Proc. Exp. Biol. Med. 212, 305–312. 4. Schoentgen, F., Metz-Boutigue, M., Jolles, J., Constans, J., and Jolles, P. (1986) Complete amino acid sequence of human vitamin D-binding protein (group specific component): Evidence of a three-fold internal homology as in serum albumin and a-fetoprotein. Biochem. Biophys. Acta 871, 189–198. 5. Viau, M., Constans, J., Debray, H., and Montreuil, J. (1983) Isolation and characterization of the O-glycan chain of the human vitamin D binding protein. Biochem. Biophys. Res. Commun. 117, 324–331. 6. Van Baelen, H., Bouillon, R., and DeMoor, P. (1980) Vitamin Dbinding protein (Gc-globulin) binds actin. J. Biol. Chem. 255, 2270–2272. 7. Haddad, J. G. (1982) Human serum binding protein for vitamin D and its metabolites (DBP): Evidence that actin is the DBP binding component in human skeletal muscle. Arch. Biochem. Biophys. 213, 538–544. 8. Williams, M. H., Van Alstyne, E. L., and Galbraith, R. M. (1988) Evidence of a novel association of unsaturated fatty acids with Gc (vitamin D-binding) protein. Biochem. Biophys. Res. Commun. 153, 1019–1024. 9. Calvo, M., and Ena, J. M. (1989) Relations between vitamin D and fatty acid binding properties of vitamin D-binding protein. Biochem. Biophys. Res. Commun. 163, 14–17.
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