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Expression and purification of recombinant cytoplasmic domain of human erythrocyte band 3 with hexahistidine tag or chitin-binding tag in Escherichia coli
Protein Expression and Purification 34 (2004) 167–175 www.elsevier.com/locate/yprep
Expression and purification of recombinant cytoplasmic domain of human erythrocyte band 3 with hexahistidine tag or chitin-binding tag in Escherichia coli Yu Ding, Weihua Jiang, Yang Su, Hanqing Zhou, and Zhihong Zhang* Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai 200433, China Received 4 August 2003, and in revised form 17 October 2003
Abstract The cytoplasmic domain of erythrocyte band 3 (cdb3) serves as a center of membrane organization in the erythrocytes by its interaction with multiple proteins including ankyrin, protein 4.1, protein 4.2, hemoglobin, and several glycolytic enzymes. In this paper, human cdb3 was cloned into three different expression vectors controlled by T7 polymerase promoter and induced with isopropyl b-D -thiogalactopyranoside in Escherichia coli. Two of the fusion proteins containing hexahistidine tag in the N-terminal or C-terminal were purified by immobilized metal affinity column chromatography. The third recombinant cdb3 containing the affinity chitin-binding tag was purified using chitin beads followed by specific self-cleavage, which released cdb3 according to the mechanism of protein splicing. The molecular weights of purified recombinant proteins were verified by mass spectrometry. The pHdependent properties of the intrinsic tryptophan fluorescence of the three kinds of recombinant cdb3 were compared with that of the cdb3 extracted from the erythrocytes, showing that there were no significant differences between them. Using pull-down assay combined with Western blot analysis, the interaction between recombinant cdb3 and protein 4.2 C3 fragment was verified. These demonstrated that the recombinant proteins were both structurally and functionally active. The typical yields of cdb3 purified with hexahistidine tag in the N-terminal, C-terminal, and cleaved from chitin bead were 10.6, 9.6, and 1.5 mg from 1 L culture medium, respectively. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Cytoplasmic domain of band 3; Protein 4.2; Erythrocyte membrane; Protein expression in Escherichia coli; Hexahistidine tag; Affinity chitin-binding tag; Pull-down assay; Intrinsic fluorescence
The erythrocyte anion transporter, band 3, is the most abundant protein of the erythrocyte membrane, comprising approximately 25% of the total membrane protein [1]. The gene that encodes band 3 (AE1) is located on chromosome 17q21-q22 and is primarily expressed in the erythrocytes and kidney [2,3]. The calculated molecular weight of unmodified band 3 from the cDNA sequence is 101,792 Da. Band 3 consists of two structurally and functionally independent domains. The C-terminal transmembrane domain, about 50 kDa, mediates the exchange of chloride for bicarbonate anions across the plasma mem-
* Corresponding author. Fax: +86-21-65650149. E-mail address: [email protected] (Z. Zhang).
1046-5928/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2003.10.019
brane. The N-terminal cytoplasmic domain (cdb3),1 of about 43 kDa, acts as an attachment site for the erythrocyte skeleton by binding ankyrin [4], protein 4.1 [5,6], and protein 4.2 [7–9], helping us to maintain the mechanical properties and integrity of the erythrocytes. Besides, cdb3 also interacts with several glycolytic enzymes [10,11], p72(syk) protein tyrosine kinase [12], hemoglobin [13], and hemichromes [14] to take part in
1 Abbreviations used: cdb3, cytoplasmic domain of band 3; His6, six consecutive histidine residues; CBD, chitin-binding domain; Ni–NTA, nickel–nitrilotriacetic acid; BCA, bicinchoninic acid; HRP, horseradish peroxidase; IPTG, isopropyl b-D -thiogalactopyranoside; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; LC/MS, liquid chromatography/mass spectrometry.
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regulating the metabolic activity and sensing the aged or abnormal erythrocytes. Band 3 mutations can result in significant changes in the shape and deformability of the erythrocytes. Southeast Asian ovalocytosis with abnormal rigid, stomatocytic erythrocytes results from the heterozygous presence of a deletion in band 3 protein [15,16]. Some band 3 mutations in the erythrocytes, especially in cdb3, destabilize erythrocyte membranes, leading to hereditary spherocytosis [15,17,18]. So it will help us much in curing the related diseases if we know more about these mutationsÕ structural basis and the abnormality of these protein–protein interactions. Although cdb3 can be purified from the erythrocytes by digesting ghosts with a-chymotrypsin [19], the product is a mixture of polypeptides with different molecular weights and the product yield is not high. So it is difficult to use such kind of samples to do experiments such as crystallization and the detailed mechanism study on the structural basis. Using antibody that specifically reacts with cdb3, one can perform immunoprecipitation experiment for searching the proteins that bind with cdb3 in the membranes [20]. But its huge size will block some specific sites of cdb3 in the formed macromolecular complex and therefore will interfere with the protein–protein interactions. So it is convenient to introduce a tag that does not interfere with the interactions to perform the pull-down assay. And more, the tag can be used to facilitate the purification and pulldown procedure, because the tag is universal, it can be detected fast, easily, and is reproducible when using commercial reagents. Finally, the tag will also be useful in studying the dynamic structure and the mechanics of protein interactions by single molecule analysis techniques. Wang et al. [21] have cloned, expressed, and purified cdb3 from Escherichia coli and the crystal structure of resolution by X-ray cdb3 has been determined at 2.6 A diffraction [22]. But the recombinant cdb3 they used does not have any tag to facilitate the study on the interaction of cdb3 with other proteins, which is the most important character of cdb3. Moreover, the positions of residues 1–54 were not determined by the X-ray diffraction because of the flexibility of this domain. Now it is commonly believed that the extreme N-terminal of cdb3 would be the sites where some other proteins bind. So the detailed mechanism of the interactions between cdb3 and other proteins is still remaining to be determined. Another shortcoming of the reported purification method using anion exchanger was the low quality of both the purity and yield as compared with the method of affinity chromatography. Hexahistidine tag is smaller than most other affinity tags, rarely interferes with protein structure or function, and rarely requires removal by protease cleavage [23]. The IMPACT-CN system [24,25] utilizes the inducible
self-cleavage activity of a protein splicing element to separate the target protein from the chitin-binding affinity tag without using a separate protease, purifying the native recombinant protein in a single chromatographic step. Building on the previous works of other groups, it should be possible to obtain recombinant cdb3 in high yields by introducing a proper tag followed by affinity chromatography. Here, we report the methods that use hexahistidine tag or affinity chitin-binding tag for expression and purification cdb3 in E. coli, and then compare the structural and functional character with that of cdb3 extracted from the erythrocytes.
Materials and methods Materials The bacterial (E. coli) host, the cloning vector pET21, Perfect Protein markers, and Western blot kits were obtained from Novagen (Madison, WI). Pfu polymerase, nucleotides, agarose gel, DNA extraction kit, and high pure PCR purification kit were purchased from Roche Diagnostics (Indianapolis, IN). Primers were synthesized at Bioasia (Shanghai, China). The DNA sequencing was performed by Bioasia (Shanghai, China). The restriction endonucleases and DNA ligation kit were purchased from Takara (Dalian, China). Ni–NTA Superflow column matrix was obtained from Qiagen (Chatsworth, CA). The cloning vector pTYB12 and chitin beads were obtained from New England Biolabs (Beverly, MA). Human bloods were obtained from the Shanghai Red Cross Blood Center. The DEAE–Sepharose CL-6B matrix, Sephadex G-50 matrix, glutathione– Sepharose 4B, and pGEX-5X-1 vector were purchased from Amersham–Pharmacia Biotech (Piscataway, NJ). BCA protein assay reagent kit, Slide-A-Lyzer mini dialysis unit, goat anti-mouse secondary antibody labeled with HRP, and SuperSignal WestPico chemiluminescent substrate were from Pierce (Rockford, IL). IPTG, DTT, EDTA, imidazole, L -glutathione (reduced form), PMSF, a-chymotrypsin, and anti-band 3 monoclonal antibody were obtained from Sigma (St. Louis, MO). Amicon Ultra-15 centrifugal filter (MWCO 10,000) was obtained from Millipore (Bedford, MA). All other reagents were of analytical grade. Cloning the cdb3 coding region into vector pT7470 and pTYB12 The pT7470 expression vector containing both N-terminal and C-terminal hexahistidine tags was constructed from plasmid pET21a. Briefly, a pair of synthesized DNA fragments containing hexahistidine sequence with a NdeI end and an EcoRI end was ligated into NdeI- and EcoRI-digested pET21a. For vector
Y. Ding et al. / Protein Expression and Purification 34 (2004) 167–175 Table 1 Sequences of synthesized oligonucleotide primers used in cloning the cdb3 into vectors pT7470 and pTYB12 Primer
Sequencea
P1 P2 P3 P4 P5 P6
50 -TAGGATCCGAGGAGCTGCAGGATGATTAT-30 50 -AACTCGAGCTAGAAGAGCTGGCCTGTCTG-30 50 -ATACATATGGAGGAGCTGCAGGAT-30 50 -AAACTCGAGGAAGAGCTGGCCTGT-30 50 -TCTCATATGGAGGAGCTGCAGGAT-30 50 -ACTCTCGAGTCAGAAGAGCTGGCCTGTCTG-30
a P1 and P2 are for vector pT7470 and encode hexahistidine tag in the N-terminal, P3 and P4 are for vector pT7470 and encode hexahistidine tag in the C-terminal, and P5 and P6 are for vector pTYB12. The sequences cut by respective restriction endonucleases are underlined.
verification and further cloning, a BamHI was introduced immediately before EcoRI. The synthesized fragments are as follows, where the sequences encoding six consecutive histidine residues are underlined. + strand: 50 -TATGCACCACCACCACCACCACG GATCCG-30 ) strand: 30 -ACGTGGTGGTGGTGGTGGTGCC TAGGCTTAA-50 Previously, we had constructed a pRSET-cdb3 plasmid derived from pHB3 plasmid (a gift of Dr. S.E. Lux, The ChildrenÕs Hospital, Boston) containing full-length human erythrocyte band 3 cDNA [26]. Now we used pRSET-cdb3 plasmid as a template to clone cdb3 into pT7470 via primers P1 and P2 (hexahistidine in N-terminal), P3 and P4 (hexahistidine in C-terminal) (Table 1). Another vector we used for expression of fusion protein chitin-binding domain (CBD)-cdb3 via primers P5 and P6 (Table 1) was pTYB12. The conditions used for both PCR amplifications were same: 94 °C for 5 min followed by 30 cycles at 94 °C for 60 s, 58 °C for 60 s, 72 °C for 90 s, and finally 72 °C for 5 min. The PCR product was purified by cutting the desired band from agarose gel and then ligated into the expression vector. For recombinant protein (N)His6-cdb3, the BamHI and XhoI sites were used. Since there was a BamHI endonuclease site in cdb3, we carried out a partial digestion. The desired long form digested PCR product was separated by gel recovery and then ligated into BamHI- and XhoI-digested pT7470 vector. For recombinant protein (C)His6-cdb3, the NdeI and XhoI sites were used. For pTYB12, the NdeI and XhoI sites were used. The ligated vector was then transformed into the host cell (E. coli DH5a). The insert was sequenced for verification purposes. Expression and purification of fusion protein (N)His6cdb3 and (C)His6-cdb3 The bacteria containing the desired clone with pT7470-cdb3 expression vector were transformed into
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HMS174 (DE3) for expression. After grown overnight at 37 °C in 20 ml LB medium supplemented with 100 lg/ ml ampicillin, a portion (8 ml) of the bacterial suspension was then transferred into 4 L fresh LB medium. Transformed HMS174 (DE3) cells were allowed to grow with shaking (240 rpm) at 37 °C to OD (600 nm) of 0.6. The expression of (N)His6-cdb3 was induced by addition of IPTG to a final concentration of 1 mM and then incubated at 30 °C for additional 4 h. For expression of (C)His6-cdb3, the IPTG concentration was same, but the incubation time was longer (6 h). The cells were collected by centrifugation at 6000g for 15 min. The harvested cells were frozen at )20 °C until use. Unless otherwise stated, all following procedures were carried out at 0–4 °C. The cells were thawed and resuspended in 200 ml lysis buffer I (50 mM Tris–HCl, pH 8.0, 200 lg/ml PMSF). Cells were lysed by sonication and cellular debris was removed by centrifugation at 20,000g for 20 min. The supernatant was then loaded on a 20 ml (10 1.6 cm) column of DEAE–Sepharose CL-6B equilibrated with 5 mM sodium phosphate buffer, pH 7.4. Then the proteins were eluted with 100 ml salt gradient of 0–1 M NaCl in 5 mM sodium phosphate buffer with the flow rate of 1 ml/min. Following SDS– PAGE assay, the fraction contained mainly cdb3 was collected and loaded on a 10 ml (15 1 cm) Ni–NTA Superflow column equilibrated with lysis buffer I containing 5 mM imidazole. The column was washed with 20 ml lysis buffer I containing 20 mM imidazole followed by elution with 10 ml lysis buffer I containing 200 mM imidazole. The peak was collected and loaded on a Sephadex G-50 column (30 1.6 cm) equilibrated with 5 mM sodium phosphate buffer, pH 7.4. The desalted protein was collected and concentrated by Amicon Ultra-15 centrifugal filter at 4000g for 20 min. An equal volume of glycerol was added to make a final glycerol concentration of 50% followed by storage at )20 °C. Expression and purification of fusion protein CBD-cdb3 The bacteria containing the desired clone with pTYB12-cdb3 expression vector were also transformed into HMS174 (DE3) for expression. The procedure was same as that for His-cdb3 before induction with IPTG. The culture medium was cooled to 25 °C before addition of IPTG to a final concentration of 0.1 mM. After additional 3 h incubation at 25 °C, the cells were collected by centrifugation at 6000g for 15 min. The harvested cells were frozen at )20 °C until use. Unless otherwise stated, all procedures were carried out at 0–4 °C. The cells were thawed and resuspended in 200 ml lysis buffer II (50 mM Tris–HCl, pH 8.0, 500 mM NaCl, and 200 lg/ml PMSF). The cells were incubated on ice for 15 min and then lysed by sonication, and the cellular debris was removed by centrifugation at 20,000g for 20 min. The supernatant was then loaded on a 20 ml
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(10 1.6 cm) column of chitin beads equilibrated with lysis buffer II. After washing the column with 200 ml lysis buffer II at the flow rate of 1 ml/min, the column was quickly flushed with 60 ml lysis buffer II containing 50 mM DTT at 20 °C and left for additional 40 h. The cleaved cdb3 was eluted with lysis buffer II and loaded on a Sephadex G-50 column (30 1.6 cm) equilibrated with 5 mM sodium phosphate buffer, pH 7.4. The desalted protein was collected and concentrated by Amicon Ultra-15 centrifugal filter at 4000g for 20 min. An equal volume of glycerol was added to make a final glycerol concentration of 50% followed by storage at )20 °C. Purification of cdb3 from the human erythrocytes Purification of cdb3 from the erythrocytes was performed according to Appell and Low [19]. Briefly, the fresh erythrocytes were washed with PBS (150 mM NaCl, 5 mM sodium phosphate buffer, pH 7.4) and then hemolyzed in 5 mM sodium phosphate buffer, pH 7.4, followed by centrifugation at 20,000g for 15 min. Following washing three times, the ghosts were incubated in 0.5 mM EDTA, pH 8.0, at 37 °C for 30 min to deplete spectrin and actin, and the ghosts were collected again by centrifugation at 20,000g for 15 min. The ankyrin, protein 4.1, and protein 6 were removed by incubating the pellets in 10 volumes of 0.17 M acetic acid for 20 min at 37 °C. The vesicles were collected by centrifugation at 20,000g for 15 min, washed in 0.1 M sodium phosphate buffer, pH 7.4, and suspended in 20 volumes of ice-cold 10 mM sodium phosphate buffer, pH 7.4. The cdb3 fragment was then cleaved from the vesicles by treatment of the vesicle suspension with 1 lg/ml a-chymotrypsin at 0 °C for 20 min. The digestion was terminated by addition of 200 lg/ml PMSF. The cdb3 was collected by centrifugation. The supernatant was collected and concentrated by Amicon Ultra-15 centrifugal filter at 4000g for 20 min. An equal volume of glycerol was added to make a final glycerol concentration of 50% followed by storage at )20 °C. SDS–PAGE and Western blot The protein samples (typically 10 ll) were mixed with 5 ll of 3 loading buffer [150 mM Tris–HCl (pH 6.8), 300 mM DTT, 6% (w/v) SDS, 0.06% (w/v) bromophenol blue, and 30% (v/v) glycerol], and boiled for 3 min prior to loading on a 10 or 12% Tris–HCl gel for electrophoresis using Bio-RadÕs Mini-PROTEIN 3 Cell. Proteins in the gel were visualized by staining with Coomassie G-250. The purity of stained proteins was determined by UVPÕs GelWorks gel analysis software and the protein concentration was determined by BCA method according to the protocol of BCA Protein Assay Reagent Kit (Pierce).
For Western blot analysis, the proteins were electrophoretically transferred onto 0.20 lm nitrocellulose transfer membrane (Schleicher & Schuell). The nitrocellulose was blocked by incubation in blocking buffer [150 mM NaCl, 50 mM Tris–HCl, pH 7.4, and 5% (w/v) low fat milk powder] for 1 h at 37 °C. Then, the membrane was incubated with anti-band 3 monoclonal antibody (Sigma) (1:5000) in the blocking buffer for 1 h at 37 °C followed by washing four times with washing buffer [150 mM NaCl, 50 mM Tris–HCl, pH 7.4, and 0.1% (v/v) Tween 20] for 10 min each wash. The membrane was then incubated with goat anti-mouse secondary antibody conjugated with HRP (1:5000) for 1 h at 37 °C. Also, the unbound HRP-conjugated secondary antibody was removed by washing with washing buffer four times for 10 min each wash. Finally, the membrane was incubated with working solution (SuperSignal WestPico Substrate) for 1 min and then pressed against an autoradiographic film. Determination of the molecular weight of recombinant proteins by mass spectrometry The purified recombinant proteins were desalted using HP 1100 LC system with column Zorbax 300SBC18. A linear gradient was formed by deionized water and 80% acetonitrile in 35 min. The eluate of peak fraction was collected and loaded on a mass spectrometer (PE API 165 LC/MS System) for measurement of the molecular weight. The pH-dependent property of the intrinsic fluorescence of recombinant cdb3 The measurement of intrinsic fluorescence of cdb3 was according to Low and co-workers [19,21]. Briefly, the purified recombinant cdb3 was dialyzed against different pH buffers containing 50 mM sodium phosphate, 50 mM sodium borate, 70 mM NaCl, and with or without 6 M urea. The protein concentration was determined by BCA method. The concentration of cdb3 in all measurement samples was 40 lg/ml. Fluorescence measurements were performed at 25 °C with a Hitachi M 850 spectrofluorometer. The wavelength of excitation was 290 nm and the fluorescence emission at 300–450 nm was detected. Expression of GST-Protein 4.2 C3 and GST The plasmid pGEX3X-Protein 4.2 C3 was a gift of Dr. J. Basu (Bose Institute, India). Protein 4.2 C3, which encompasses amino acid residues 187–260 of protein 4.2, is a critical domain for interaction of protein 4.2 with cdb3 [9]. The plasmid was transformed into E. coli HMS174 (DE3) and the expression was carried out in the presence of 0.1 mM IPTG at 25 °C for 3 h. The cells
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were collected, suspended in lysis buffer III (50 mM Tris–HCl, pH 8.0, 5 mM EDTA, 50 mM NaCl, 1% (v/v) Triton X-100, and 200 lg/ml PMSF), and sonicated on ice bath. The supernatant was collected by centrifugation at 20,000g for 20 min, loaded on a glutathione–Sepharose 4B column (5 0.7 cm), and washed with Tris–HCl buffer, pH 8.0, containing 1% (v/v) Triton X-100. Then, the protein was eluted with elution buffer (50 mM glutathione in Tris–HCl buffer, pH 8.0, and 1% (v/v) Triton X-100). Finally, the eluate was loaded on a Sephadex G50 column (30 1.6 cm) equilibrated with 5 mM sodium phosphate buffer, pH 7.4, to desalt. The desalted protein was collected and concentrated by Amicon Ultra-15 centrifugal filter at 4000g for 20 min. An equal volume of glycerol was added to make a final glycerol concentration of 50% followed by storage at )20 °C. The recombinant protein GST was expressed using pGEX-5X-1 vector and the same was purified as recombinant protein GST-Protein 4.2 C3. Pull-down assay The purified GST-Protein 4.2 C3 protein (10 lg) or control GST protein (8 lg) was loaded on the glutathione–Sepharose 4B resin (100 ll of 50% slurry) balanced with PBS containing 0.1% (v/v) Triton X-100. Hundred micrograms of the target protein [(N)His6-cdb3, (C)His6-cdb3, cleaved CBD-cdb3 or native cdb3 purified from the erythrocytes] was mixed with BSA (100 lg) and loaded on the resin (total volume 200 ll, 200 lg/ml PMSF). The mixture was loaded on Spin X Cups (Seize X Immunoprecipitation Kit, Pierce) by gently shaking overnight at 4 °C and centrifuged at 2000g for 30 s. After washing the resin four times with PBS containing 0.1% (v/v) Triton X-100, the proteins were eluted by SDS–PAGE loading buffer. The eluate was analyzed by SDS–PAGE followed with Western blot.
Results and discussion Cloning of the cdb3 to vectors pT470 and pTYB12 Both the pT7470 and pTYB12 vectors contain T7 polymerase promoter and can be induced by IPTG. For recombinant protein (N)His6-cdb3, the cdb3 gene was amplified with primers that introduced a BamHI site at its 50 end and a stop codon followed by a XhoI site at its 30 end. The first methionine residue encoded by the gene was not included because the vector pT7470 already contained it. It was verified by DNA sequencing that the expressed fusion protein (N)His6-cdb3 consists of 387 residues, compared with original cdb3 residues 1–379, due to additional eight residues (His–His–His–His–HisHis–Gly–Ser) inserted between the N-terminal first me-
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thionine residue and second glycine residue. For recombinant protein (C)His6-cdb3, the cdb3 gene was amplified with primers with a NdeI site at its 50 end and a XhoI site at its 30 end. It was verified by DNA sequencing that the expressed fusion protein (C)His6-cdb3 consists of 387 residues, compared with original cdb3 residues 1–379, due to additional eight residues (Leu–Glu–His–His–His–His–His–His) inserted in the C-terminal. For plasmid pTYB12, the cdb3 gene was amplified with primers that introduced a NdeI site at its 50 end and a stop codon followed by a XhoI site at its 30 end. The expressed protein has additional three residues (Ala–Gly–His) attached to the N-terminal after the affinity chitin-binding tag was self-cleaved, which was also verified by DNA sequencing. Expression and purification of cdb3 with hexahistidine tag To optimize the expression conditions, first, we tested the influence of temperature. It was observed that when induction time was 4 h, the expression of His6-cdb3 at 30 °C was higher than at 37 or 25 °C, and in all tested temperatures, most of the His6-cdb3 was in soluble form. Second, we tested the influence of IPTG concentration (0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, and 5 mM) and found that the yield was linear to IPTG below 1 mM concentration, but when IPTG concentration exceeded 1 mM, the yield did not increase significantly. Finally, we found that when induction time exceeded 4 h under the condition of 1 mM IPTG and 30 °C, the (N)His6cdb3 yield (about 30 mg from 1 L culture medium) did not increase, but (C)His6-cdb3 still increased up to about 6 h. So all further expression of His6-cdb3 was conducted using 1 mM IPTG at a growth temperature of 30 °C, the inducing time of (N)His6-cdb3 and (C)His6cdb3 was 4 and 6 h, respectively. We have tried to purify the His6-cdb3 protein by directly loading the lysate on the Ni–NTA Superflow resin, but there were always some contaminating proteins after elution (using this one-step method, the purity of His6-cdb3 was about 95% by SDS–PAGE analysis), no matter increasing the concentration of imidazole in the elution buffer, prolonging the wash time or using gradient elution. For increase in the purity of the recombinant protein, an anion exchanger step was added. The His6-cdb3 then was eluted from a DEAE– Sepharose CL-6B column at the NaCl concentration about 0.4 M. For desalinization, the method of dialysis was not desirable because it was time consuming and some of the proteins were easy to degrade during dialysis though the operation was performed at 4 °C. Therefore, we used gel filter instead. The final purity of His6-cdb3 was above 98% by SDS–PAGE assay. We have observed that after desalinization, the 43 kDa His6-cdb3 protein became unstable and easy to degrade to a 41 kDa fragment. So,
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it was immediately ultrafiltered and then an equal volume of glycerol was added to make a final glycerol concentration of 50% and stored at )20 °C to avoid degradation. Expression and purification of cdb3 with affinity chitinbinding tag We found that the majority of the expressed fusion protein CBD-cdb3 induced by IPTG was in the inclusion body by SDS–PAGE analysis. In an attempt to increase the yield of soluble and active CBD-cdb3, several experiments using different induction temperatures were carried out. When the temperature was decreased from 37 to 25 °C, the soluble fraction was increased, though not significantly. To choose the induction time it was found that 3 h was enough and longer induction did not result in improvement of soluble fraction. For impact of IPTG under condition of 25 °C and induction for 3 h, the final concentrations of IPTG from 0.02 to 5 mM were tested. The result showed that 0.1 mM IPTG was enough to give the highest expression of soluble CBDcdb3 (data not shown). The yield of soluble CBD-cdb3 was about 1.5 mg from 1 L culture medium, which was enough for further analysis of protein–protein interaction. So the optimization condition we chose was induction at 25 °C for 3 h with 0.1 mM IPTG. We used the inducible self-cleavage activity of the protein splicing element in CBD tag to separate the target-cleaved cdb3 protein (43 kDa) in the C-terminal region of the fusion protein from the chitin-binding affinity tag (58 kDa). The self-cleavage procedure does need not other expensive proteases and it happens just under high thiol (such as DTT, b-mercaptoethanol or cysteine) circumstances. After reaction with thiol reagent, the N-terminal is first cleaved and then the Cterminal region is cleaved [24,25]. After cleavage, the CBD fragment still bound with the chitin bead, the cleaved cdb3 protein was in the eluted cleavage buffer. During the self-cleavage testing, if the NaCl concentration was lower than 100 mM, most of the CBD-cdb3 was not spliced and was still bound to the chitin bead as 101 kDa fusion protein. Meanwhile, the protein should be exposed to room temperature for 40 h, which would increase the possibility of the degradation of recombinant cdb3. However, we found that when there were high salt (0.5 M NaCl) and high thiol (50 mM DTT), the protein was stable during purification. Because the purification depends on thiol-induced intein self-cleavage mechanism, the eluate after self-cleavage contained not only cleaved recombinant cdb3, but also a small peptide of 15 residues which had a molecular weight of about 1.6 kDa [25]. The result by use of Sephadex G-50 resin for further purification showed that CBD-cdb3 was efficiently separated from this small peptide and the high concentration salt in elution buffer.
Verification of purification by SDS–PAGE, Western blot, and mass spectrometry Figs. 1–3 show the typical SDS–PAGE and Western blot analysis for the purification of (N)His6-cdb3, (C)His6-cdb3, and CBD-cdb3, respectively. The results showed that the leakage expression can be neglected before IPTG induction, so it was not necessary to test the strain pLysS or pLysE. Lane 6 in Fig. 1 was the Western blot result of induced bacteria with band 3 antibody. A faint band about 31 kDa can been seen, which was the degraded cdb3 during induction. The degraded cdb3 can be efficiently separated from the fulllength recombinant cdb3 by DEAE anion exchanger. There was a minor leakage expression of (C)His6-cdb3 in unduced bacteria (Fig. 2, lane 7), but it did not interfere with the latter purification. There were some degraded fusion proteins when pTYB12-cdb3 plasmid was induced (Fig. 3, lane 10). But most of them were in the inclusion body and did not appear in the soluble CBD-cdb3 fraction (Fig. 3, lane 11). Tables 2–4 summarize the results of purification. The purity of stained proteins was determined by UVPÕs GelWorks gel analysis software and the concentration of proteins was determined by BCA method. The typical yields of (N)His6-cdb3, (C)His6-cdb3, and CBDcdb3 were about 10.6, 9.6, and 1.5 mg from 1 L culture medium, respectively. The calculated molecular weights of (N)His6-cdb3, (C)His6-cdb3, and the cdb3 cleaved from CBD-cdb3 are
Fig. 1. SDS–PAGE and Western blot analysis of (N)His6-cdb3. Lane 1, uninduced bacterial lysate; lane 2, induced bacterial lysate; lane 3, eluate from DEAE–Sepharose CL-6B anion exchanger; and lane 4, eluate from Ni–NTA resin purification. Lanes 5–8 represent the Western blot result of lanes 1–5 using anti-band 3 antibody.
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Table 2 Purification of recombinant (N)His6-cdb3 from an E. coli expression systema
Supernatant Anion exchanger Ni–NTA Gel filtration a
Total protein (mg)
(N) His6-cdb3 (mg)
Fold enrichment
Relative yield
86.5 20.6 13.0 10.8
27.7 19.0 12.7 10.6
1 2.9 3.0 3.1
1 0.69 0.46 0.38
From 1 L of E. coli culture medium.
Table 3 Purification of recombinant (C)His6-cdb3 from an E. coli expression systema
Fig. 2. SDS–PAGE and Western blot analysis of (C)His6-cdb3. Lane 1, NovagenÕs Perfect Protein markers; lane 2, uninduced bacterial lysate; lane 3, induced bacterial lysate; lane 4, eluate from DEAE– Sepharose CL-6B anion exchanger; and lane 5, eluate from Ni–NTA resin purification. Lanes 6–10 represent the Western blot result of lanes 1–5 using anti-band 3 antibody. S-Protein HRP conjugate in the Perfect Protein Western blot kits was used to detect the NovagenÕs Perfect Protein markers in Western blot analysis.
Supernatant Anion exchanger Ni–NTA Gel filtration a
Total protein (mg)
(C) His6-cdb3 (mg)
Fold enrichment
Relative yield
121 25.7 14.9 9.8
32.7 23.4 14.6 9.6
1 3.4 3.6 3.6
1 0.72 0.45 0.29
From 1 L of E. coli culture medium.
Table 4 Purification of recombinant CBD-cdb3 from an E. coli expression systema Total CBD-cdb3 protein (mg) (mg) Supernatant Chitin beads Gel filtration
117 2.13 1.57
Folder enrichment
2.52 (5.85)b 1 2.05 44.7 1.54 45.5
Relative yield 1 0.81 0.61
a
From 1 L of E. coli culture medium. The value was calculated according to the protein molecular weight before (101 kDa) and after (43 kDa) self-cleavage; the value in parentheses was the origin data gained from gel band density of 101 kDa CBD-cdb3 protein. b
on the measurement. They all accorded with the calculated molecular weights from the respective DNA sequences. Fig. 3. SDS–PAGE and Western blot analysis of CBD-cdb3. Lane 1, cdb3 purified from erythrocyte ghosts by digestion with a-chymotrypsin; lane 2, NovagenÕs Perfect Protein markers; lane 3, uninduced bacterial lysate; lane 4, induced bacterial lysate; lane 5, the proteins that bound with chitin beads after washing; and lane 6, eluate of selfcleaved proteins. Lanes 7–12 represent the Western blot result of lanes 1–6 using anti-band 3 antibody. S-Protein HRP conjugate in the Perfect Protein Western blot kits was used for detecting the NovagenÕs Perfect Protein markers in Western blot analysis.
43502.68, 43600.80, and 42,800.98 Da, respectively, by ExPASyÕs Peptide Mass Program. The two purified proteins were verified by mass spectrometry (Fig. 4, (C)His6-cdb3 was not tested). The molecular weights of (N)His6-cdb3 and the cdb3 cleaved from CBD-cdb3 were 43,501.0 Da, and 42,805.0 Da, respectively, based
The intrinsic fluorescence of the recombinant cdb3 One of the most important characters of the cdb3 protein is its ability to undergo a large and fully reversible pH-dependent conformational change [27]. The intrinsic fluorescence emission spectrum is one of the convenient ways to monitor this kind of structural change. There are four tryptophan residues in the cdb3 protein. In our experiment, the intrinsic fluorescence spectra were mainly contributed by these tryptophan residues. It is well known that both the fluorescence intensity and emission peak will be changed if the surrounding of the tryptophan residue changes. Especially, the fluorescence intensity will be decreased after tryptophan residue moves from hydrophilic
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Fig. 4. Verification of the recombinant cdb3 by mass spectrometry. (Left) (N)His6-cdb3 fusion protein and (right) the cdb3 cleaved from CBD-cdb3 fusion protein.
environment to hydrophobic environment. We have tested the recombinant proteins (N)His6-cdb3, (C)His6cdb3, and cleaved CBD-cdb3, found that the intrinsic fluorescence increased significantly when the pH increased, and meanwhile the emission peak moved to a longer wavelength. The shapes of the fluorescence spectrum among these three recombinant cdb3 and the
cdb3 purified from the human erythrocytes were similar to each other, especially at higher pH (see Fig. 5). When denatured in 6 M urea, the fluorescence emission peaks moved to about 350 nm and the pH dependence of the intrinsic fluorescence disappeared (data not shown). The character of the pH dependence of the intrinsic fluorescence demonstrated that the recombinant proteins were structural active. Pull-down assay Protein 4.2 is a peripheral membrane protein that presents in 200,000 copies per erythrocyte and comprises approximately 5% of the total membrane proteins of the erythrocyte. It is commonly believed that the interaction between band 3, ankyrin, and protein 4.2 is one of the most important linkages between the skeleton network and the plasma membrane in the erythrocytes, although the information on the binding site of protein 4.2 on cdb3 is limited [22]. C3 is a protein 4.2 fragment of residues 187–260, a domain critical for the protein 4.2–cdb3 interaction [9]. Using pull-down assay, the ability of interaction between GST-Protein 4.2 C3 and cdb3 was studied
Fig. 5. pH dependence of the intrinsic fluorescence of recombinant cdb3. The relative intensity of the fluorescence emission from 300 to 450 nm was plotted (the excitation wavelength was 290 nm). The emission spectra of the recombinant proteins were obtained at pH values of 10.0, 9.0, 8.0, 7.0, and 6.0 (upper to lower). (A) The cdb3 purified from the human erythrocytes; (B) the cdb3 cleaved from CBDcdb3 fusion protein; (C) (N)His6-cdb3 fusion protein; and (D) (C)His6-cdb3 fusion protein.
Fig. 6. Association of recombinant cdb3 with protein 4.2. The purified GST-Protein 4.2 C3 (lanes 3, 5, 7, and 9) or control GST protein (lanes 2, 4, 6, and 8) was loaded on the glutathione–Sepharose 4B resin balanced with PBS containing 0.1% (v/v) Triton X-100. The target protein, cdb3 purified from erythrocyte (lanes 2 and 3), (N)His6-cdb3 (lanes 4 and 5), (C)His6-cdb3 (lanes 6 and 7), or cleaved CBD-cdb3 (lanes 8 and 9), was loaded on the resin by gently shaking overnight at 4 °C. After washing, the proteins eluted by SDS–PAGE loading buffer were analyzed by SDS–PAGE followed with Western blot using antiband 3 antibody. Lane 1, NovagenÕs Perfect Protein markers.
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here. Both (N)His6-cdb3, (C)His6-cdb3, and CBD-cdb3 can react with protein 4.2 C3 fragment (Fig. 6). Using purified GST as a control, it was observed that GST could not pull (N)His6-cdb3, (C)His6-cdb3 or CBD-cdb3 down. The pull-down assay to some extent verified that the recombinant proteins were functionally active. In summary, we have developed novel methods to express cdb3 with tags for further research, especially in protein–protein interaction and crystallography study. All the three recombinant cdb3 were both structurally and functionally similar to the native cdb3. The yields of cdb3 with hexahistidine tag in N- or C-terminal were similar and much higher than CBD-cdb3. The purification procedures were timesaving and suitable for getting enough homogeneous proteins with high purity for further research. Meanwhile, cleaved CBD-cdb3 was much more stable than His6-cdb3 after long time preservation. Acknowledgments The work was supported by Grant 2001CB5102 from the National Basic Research Priorities Program of China and the grants (30070205 and 30370380) from the National Natural Science Foundation of China. The authors thanks Yi Zhang at the Research Center of Biotechnology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, for advice on construction of the vector pT7470. References [1] P.S. Low, Structure and function of the cytoplasmic domain of band 3: center of erythrocyte membrane-peripheral protein interactions, Biochim. Biophys. Acta 864 (1986) 145–167. [2] S.E. Lux, K.M. John, R.R. Kopito, H.F. Lodish, Cloning and characterization of band 3, the human erythrocyte anionexchange protein (AE1), Proc. Natl. Acad. Sci. USA 86 (1989) 9089–9093. [3] C.C. Wang, R. Moriyama, C.R. Lombardo, P.S. Low, Partial characterization of the cytoplasmic domain of human kidney band 3, J. Biol. Chem. 270 (1995) 17892–17897. [4] V. Bennett, P.J. Stenbuck, Association between ankyrin and the cytoplasmic domain of band 3 isolated from the human erythrocyte membrane, J. Biol. Chem. 255 (1980) 6424–6432. [5] C.R. Lombardo, B.M. Willardson, P.S. Low, Localization of the protein 4.1-binding site on the cytoplasmic domain of erythrocyte membrane band 3, J. Biol. Chem. 267 (1992) 9540–9546. [6] J.C. Pinder, A. Chung, M.E. Reid, W.B. Gratzer, Membrane attachment sites for the membrane cytoskeletal protein 4.1 of the red blood cell, Blood 82 (1993) 3482–3488. [7] C. Korsgren, C.M. Cohen, Associations of human erythrocyte band 4.2. Binding to ankyrin and to the cytoplasmic domain of band 3, J. Biol. Chem. 263 (1988) 10212–10218. [8] A.C. Rybicki, S. Musto, R.S. Schwartz, Identification of a band-3 binding site near the N-terminus of erythrocyte membrane protein 4.2, Biochem. J. 309 (Pt 2) (1995) 677–681. [9] R. Bhattacharyya, A.K. Das, P.K. Moitra, B. Pal, I. Mandal, J. Basu, Mapping of a palmitoylatable band 3-binding domain of human erythrocyte membrane protein 4.2, Biochem. J. 340 (Pt 2) (1999) 505–512.
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[10] E. Huber, H.G. Baumert, G. Spatz-Kumbel, D. Schubert, Associations between erythrocyte band 3 protein and aldolase in detergent solution: determining their stoichiometry by analytical ultracentrifugation, Eur. J. Biochem. 242 (1996) 293–300. [11] B. von Ruckmann, D. Schubert, The complex of band 3 protein of the human erythrocyte membrane and glyceraldehyde-3-phosphate dehydrogenase: stoichiometry and competition by aldolase, Biochim. Biophys. Acta 1559 (2002) 43–55. [12] M.W. Musch, E.M. Hubert, L. Goldstein, Volume expansion stimulates p72(syk) and p56(lyn) in skate erythrocytes, J. Biol. Chem. 274 (1999) 7923–7928. [13] J.A. Walder, R. Chatterjee, T.L. Steck, P.S. Low, G.F. Musso, E.T. Kaiser, P.H. Rogers, A. Arnone, The interaction of hemoglobin with the cytoplasmic domain of band 3 of the human erythrocyte membrane, J. Biol. Chem. 259 (1984) 10238–10246. [14] S.M. Waugh, P.S. Low, Hemichrome binding to band 3: nucleation of Heinz bodies on the erythrocyte membrane, Biochemistry 24 (1985) 34–39. [15] P. Jarolim, J. Palek, H.L. Rubin, J.T. Prchal, C. Korsgren, C.M. Cohen, Band 3 Tuscaloosa: Pro327–Arg327 substitution in the cytoplasmic domain of erythrocyte band 3 protein associated with spherocytic hemolytic anemia and partial deficiency of protein 4.2, Blood 80 (1992) 523–529. [16] M.J. Tanner, L. Bruce, P.G. Martin, D.M. Rearden, G.L. Jones, Melanesian hereditary ovalocytes have a deletion in red cell band 3, Blood 78 (1991) 2785–2786. [17] P.B. Jenkins, G.K. Abou-Alfa, D. Dhermy, E. Bursaux, C. Feo, A.L. Scarpa, S.E. Lux, M. Garbarz, B.G. Forget, P.G. Gallagher, A nonsense mutation in the erythrocyte band 3 gene associated with decreased mRNA accumulation in a kindred with dominant hereditary spherocytosis, J. Clin. Invest. 97 (1996) 373–380. [18] N. Alloisio, P. Maillet, G. Carre, P. Texier, A. Vallier, F. Baklouti, N. Philippe, J. Delaunay, Hereditary spherocytosis with band 3 deficiency. Association with a nonsense mutation of the band 3 gene (allele Lyon), and aggravation by a low-expression allele occurring in trans (allele Genas), Blood 88 (1996) 1062–1069. [19] K.C. Appell, P.S. Low, Partial structural characterization of the cytoplasmic domain of the erythrocyte membrane protein, band 3, J. Biol. Chem. 256 (1981) 11104–11111. [20] B.M. Willardson, B.J. Thevenin, M.L. Harrison, W.M. Kuster, M.D. Benson, P.S. Low, Localization of the ankyrin-binding site on erythrocyte membrane protein, band 3, J. Biol. Chem. 264 (1989) 15893–15899. [21] C.C. Wang, J.A. Badylak, S.E. Lux, R. Moriyama, J.E. Dixon, P.S. Low, Expression, purification, and characterization of the functional dimeric cytoplasmic domain of human erythrocyte band 3 in Escherichia coli, Protein Sci. 1 (1992) 1206–1214. [22] D. Zhang, A. Kiyatkin, J.T. Bolin, P.S. Low, Crystallographic structure and functional interpretation of the cytoplasmic domain of erythrocyte membrane band 3, Blood 96 (2000) 2925–2933. [23] J. Crowe, B.S. Masone, J. Ribbe, One-step purification of recombinant proteins with the 6 His tag and Ni–NTA resin, Mol. Biotechnol. 4 (1995) 247–258. [24] M.Q. Xu, M.W. Southworth, F.B. Mersha, L.J. Hornstra, F.B. Perler, In vitro protein splicing of purified precursor and the identification of a branched intermediate, Cell 75 (1993) 1371–1377. [25] S. Chong, Y. Shao, H. Paulus, J. Benner, F.B. Perler, M.Q. Xu, Protein splicing involving the Saccharomyces cerevisiae VMA intein. The steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing system, J. Biol. Chem. 271 (1996) 22159–22168. [26] Z. Li, F. Peng, C.N. Ji, C.L. Huan, Z.H. Zhang, Cloning and expression of the cytoplasmic domain of human erythrocyte band 3 protein, Prog. Biochem. Biophys. 26 (1999) 492–494. [27] J. Zhou, P.S. Low, Characterization of the reversible conformational equilibrium in the cytoplasmic domain of human erythrocyte membrane band 3, J. Biol. Chem. 276 (2001) 38147–38151.
Expression and purification of recombinant cytoplasmic domain of human erythrocyte band 3 with hexahistidine tag or chitin-binding tag in Escherichia coli Yu Ding, Weihua Jiang, Yang Su, Hanqing Zhou, and Zhihong Zhang* Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai 200433, China Received 4 August 2003, and in revised form 17 October 2003
Abstract The cytoplasmic domain of erythrocyte band 3 (cdb3) serves as a center of membrane organization in the erythrocytes by its interaction with multiple proteins including ankyrin, protein 4.1, protein 4.2, hemoglobin, and several glycolytic enzymes. In this paper, human cdb3 was cloned into three different expression vectors controlled by T7 polymerase promoter and induced with isopropyl b-D -thiogalactopyranoside in Escherichia coli. Two of the fusion proteins containing hexahistidine tag in the N-terminal or C-terminal were purified by immobilized metal affinity column chromatography. The third recombinant cdb3 containing the affinity chitin-binding tag was purified using chitin beads followed by specific self-cleavage, which released cdb3 according to the mechanism of protein splicing. The molecular weights of purified recombinant proteins were verified by mass spectrometry. The pHdependent properties of the intrinsic tryptophan fluorescence of the three kinds of recombinant cdb3 were compared with that of the cdb3 extracted from the erythrocytes, showing that there were no significant differences between them. Using pull-down assay combined with Western blot analysis, the interaction between recombinant cdb3 and protein 4.2 C3 fragment was verified. These demonstrated that the recombinant proteins were both structurally and functionally active. The typical yields of cdb3 purified with hexahistidine tag in the N-terminal, C-terminal, and cleaved from chitin bead were 10.6, 9.6, and 1.5 mg from 1 L culture medium, respectively. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Cytoplasmic domain of band 3; Protein 4.2; Erythrocyte membrane; Protein expression in Escherichia coli; Hexahistidine tag; Affinity chitin-binding tag; Pull-down assay; Intrinsic fluorescence
The erythrocyte anion transporter, band 3, is the most abundant protein of the erythrocyte membrane, comprising approximately 25% of the total membrane protein [1]. The gene that encodes band 3 (AE1) is located on chromosome 17q21-q22 and is primarily expressed in the erythrocytes and kidney [2,3]. The calculated molecular weight of unmodified band 3 from the cDNA sequence is 101,792 Da. Band 3 consists of two structurally and functionally independent domains. The C-terminal transmembrane domain, about 50 kDa, mediates the exchange of chloride for bicarbonate anions across the plasma mem-
* Corresponding author. Fax: +86-21-65650149. E-mail address: [email protected] (Z. Zhang).
1046-5928/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2003.10.019
brane. The N-terminal cytoplasmic domain (cdb3),1 of about 43 kDa, acts as an attachment site for the erythrocyte skeleton by binding ankyrin [4], protein 4.1 [5,6], and protein 4.2 [7–9], helping us to maintain the mechanical properties and integrity of the erythrocytes. Besides, cdb3 also interacts with several glycolytic enzymes [10,11], p72(syk) protein tyrosine kinase [12], hemoglobin [13], and hemichromes [14] to take part in
1 Abbreviations used: cdb3, cytoplasmic domain of band 3; His6, six consecutive histidine residues; CBD, chitin-binding domain; Ni–NTA, nickel–nitrilotriacetic acid; BCA, bicinchoninic acid; HRP, horseradish peroxidase; IPTG, isopropyl b-D -thiogalactopyranoside; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; LC/MS, liquid chromatography/mass spectrometry.
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regulating the metabolic activity and sensing the aged or abnormal erythrocytes. Band 3 mutations can result in significant changes in the shape and deformability of the erythrocytes. Southeast Asian ovalocytosis with abnormal rigid, stomatocytic erythrocytes results from the heterozygous presence of a deletion in band 3 protein [15,16]. Some band 3 mutations in the erythrocytes, especially in cdb3, destabilize erythrocyte membranes, leading to hereditary spherocytosis [15,17,18]. So it will help us much in curing the related diseases if we know more about these mutationsÕ structural basis and the abnormality of these protein–protein interactions. Although cdb3 can be purified from the erythrocytes by digesting ghosts with a-chymotrypsin [19], the product is a mixture of polypeptides with different molecular weights and the product yield is not high. So it is difficult to use such kind of samples to do experiments such as crystallization and the detailed mechanism study on the structural basis. Using antibody that specifically reacts with cdb3, one can perform immunoprecipitation experiment for searching the proteins that bind with cdb3 in the membranes [20]. But its huge size will block some specific sites of cdb3 in the formed macromolecular complex and therefore will interfere with the protein–protein interactions. So it is convenient to introduce a tag that does not interfere with the interactions to perform the pull-down assay. And more, the tag can be used to facilitate the purification and pulldown procedure, because the tag is universal, it can be detected fast, easily, and is reproducible when using commercial reagents. Finally, the tag will also be useful in studying the dynamic structure and the mechanics of protein interactions by single molecule analysis techniques. Wang et al. [21] have cloned, expressed, and purified cdb3 from Escherichia coli and the crystal structure of resolution by X-ray cdb3 has been determined at 2.6 A diffraction [22]. But the recombinant cdb3 they used does not have any tag to facilitate the study on the interaction of cdb3 with other proteins, which is the most important character of cdb3. Moreover, the positions of residues 1–54 were not determined by the X-ray diffraction because of the flexibility of this domain. Now it is commonly believed that the extreme N-terminal of cdb3 would be the sites where some other proteins bind. So the detailed mechanism of the interactions between cdb3 and other proteins is still remaining to be determined. Another shortcoming of the reported purification method using anion exchanger was the low quality of both the purity and yield as compared with the method of affinity chromatography. Hexahistidine tag is smaller than most other affinity tags, rarely interferes with protein structure or function, and rarely requires removal by protease cleavage [23]. The IMPACT-CN system [24,25] utilizes the inducible
self-cleavage activity of a protein splicing element to separate the target protein from the chitin-binding affinity tag without using a separate protease, purifying the native recombinant protein in a single chromatographic step. Building on the previous works of other groups, it should be possible to obtain recombinant cdb3 in high yields by introducing a proper tag followed by affinity chromatography. Here, we report the methods that use hexahistidine tag or affinity chitin-binding tag for expression and purification cdb3 in E. coli, and then compare the structural and functional character with that of cdb3 extracted from the erythrocytes.
Materials and methods Materials The bacterial (E. coli) host, the cloning vector pET21, Perfect Protein markers, and Western blot kits were obtained from Novagen (Madison, WI). Pfu polymerase, nucleotides, agarose gel, DNA extraction kit, and high pure PCR purification kit were purchased from Roche Diagnostics (Indianapolis, IN). Primers were synthesized at Bioasia (Shanghai, China). The DNA sequencing was performed by Bioasia (Shanghai, China). The restriction endonucleases and DNA ligation kit were purchased from Takara (Dalian, China). Ni–NTA Superflow column matrix was obtained from Qiagen (Chatsworth, CA). The cloning vector pTYB12 and chitin beads were obtained from New England Biolabs (Beverly, MA). Human bloods were obtained from the Shanghai Red Cross Blood Center. The DEAE–Sepharose CL-6B matrix, Sephadex G-50 matrix, glutathione– Sepharose 4B, and pGEX-5X-1 vector were purchased from Amersham–Pharmacia Biotech (Piscataway, NJ). BCA protein assay reagent kit, Slide-A-Lyzer mini dialysis unit, goat anti-mouse secondary antibody labeled with HRP, and SuperSignal WestPico chemiluminescent substrate were from Pierce (Rockford, IL). IPTG, DTT, EDTA, imidazole, L -glutathione (reduced form), PMSF, a-chymotrypsin, and anti-band 3 monoclonal antibody were obtained from Sigma (St. Louis, MO). Amicon Ultra-15 centrifugal filter (MWCO 10,000) was obtained from Millipore (Bedford, MA). All other reagents were of analytical grade. Cloning the cdb3 coding region into vector pT7470 and pTYB12 The pT7470 expression vector containing both N-terminal and C-terminal hexahistidine tags was constructed from plasmid pET21a. Briefly, a pair of synthesized DNA fragments containing hexahistidine sequence with a NdeI end and an EcoRI end was ligated into NdeI- and EcoRI-digested pET21a. For vector
Y. Ding et al. / Protein Expression and Purification 34 (2004) 167–175 Table 1 Sequences of synthesized oligonucleotide primers used in cloning the cdb3 into vectors pT7470 and pTYB12 Primer
Sequencea
P1 P2 P3 P4 P5 P6
50 -TAGGATCCGAGGAGCTGCAGGATGATTAT-30 50 -AACTCGAGCTAGAAGAGCTGGCCTGTCTG-30 50 -ATACATATGGAGGAGCTGCAGGAT-30 50 -AAACTCGAGGAAGAGCTGGCCTGT-30 50 -TCTCATATGGAGGAGCTGCAGGAT-30 50 -ACTCTCGAGTCAGAAGAGCTGGCCTGTCTG-30
a P1 and P2 are for vector pT7470 and encode hexahistidine tag in the N-terminal, P3 and P4 are for vector pT7470 and encode hexahistidine tag in the C-terminal, and P5 and P6 are for vector pTYB12. The sequences cut by respective restriction endonucleases are underlined.
verification and further cloning, a BamHI was introduced immediately before EcoRI. The synthesized fragments are as follows, where the sequences encoding six consecutive histidine residues are underlined. + strand: 50 -TATGCACCACCACCACCACCACG GATCCG-30 ) strand: 30 -ACGTGGTGGTGGTGGTGGTGCC TAGGCTTAA-50 Previously, we had constructed a pRSET-cdb3 plasmid derived from pHB3 plasmid (a gift of Dr. S.E. Lux, The ChildrenÕs Hospital, Boston) containing full-length human erythrocyte band 3 cDNA [26]. Now we used pRSET-cdb3 plasmid as a template to clone cdb3 into pT7470 via primers P1 and P2 (hexahistidine in N-terminal), P3 and P4 (hexahistidine in C-terminal) (Table 1). Another vector we used for expression of fusion protein chitin-binding domain (CBD)-cdb3 via primers P5 and P6 (Table 1) was pTYB12. The conditions used for both PCR amplifications were same: 94 °C for 5 min followed by 30 cycles at 94 °C for 60 s, 58 °C for 60 s, 72 °C for 90 s, and finally 72 °C for 5 min. The PCR product was purified by cutting the desired band from agarose gel and then ligated into the expression vector. For recombinant protein (N)His6-cdb3, the BamHI and XhoI sites were used. Since there was a BamHI endonuclease site in cdb3, we carried out a partial digestion. The desired long form digested PCR product was separated by gel recovery and then ligated into BamHI- and XhoI-digested pT7470 vector. For recombinant protein (C)His6-cdb3, the NdeI and XhoI sites were used. For pTYB12, the NdeI and XhoI sites were used. The ligated vector was then transformed into the host cell (E. coli DH5a). The insert was sequenced for verification purposes. Expression and purification of fusion protein (N)His6cdb3 and (C)His6-cdb3 The bacteria containing the desired clone with pT7470-cdb3 expression vector were transformed into
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HMS174 (DE3) for expression. After grown overnight at 37 °C in 20 ml LB medium supplemented with 100 lg/ ml ampicillin, a portion (8 ml) of the bacterial suspension was then transferred into 4 L fresh LB medium. Transformed HMS174 (DE3) cells were allowed to grow with shaking (240 rpm) at 37 °C to OD (600 nm) of 0.6. The expression of (N)His6-cdb3 was induced by addition of IPTG to a final concentration of 1 mM and then incubated at 30 °C for additional 4 h. For expression of (C)His6-cdb3, the IPTG concentration was same, but the incubation time was longer (6 h). The cells were collected by centrifugation at 6000g for 15 min. The harvested cells were frozen at )20 °C until use. Unless otherwise stated, all following procedures were carried out at 0–4 °C. The cells were thawed and resuspended in 200 ml lysis buffer I (50 mM Tris–HCl, pH 8.0, 200 lg/ml PMSF). Cells were lysed by sonication and cellular debris was removed by centrifugation at 20,000g for 20 min. The supernatant was then loaded on a 20 ml (10 1.6 cm) column of DEAE–Sepharose CL-6B equilibrated with 5 mM sodium phosphate buffer, pH 7.4. Then the proteins were eluted with 100 ml salt gradient of 0–1 M NaCl in 5 mM sodium phosphate buffer with the flow rate of 1 ml/min. Following SDS– PAGE assay, the fraction contained mainly cdb3 was collected and loaded on a 10 ml (15 1 cm) Ni–NTA Superflow column equilibrated with lysis buffer I containing 5 mM imidazole. The column was washed with 20 ml lysis buffer I containing 20 mM imidazole followed by elution with 10 ml lysis buffer I containing 200 mM imidazole. The peak was collected and loaded on a Sephadex G-50 column (30 1.6 cm) equilibrated with 5 mM sodium phosphate buffer, pH 7.4. The desalted protein was collected and concentrated by Amicon Ultra-15 centrifugal filter at 4000g for 20 min. An equal volume of glycerol was added to make a final glycerol concentration of 50% followed by storage at )20 °C. Expression and purification of fusion protein CBD-cdb3 The bacteria containing the desired clone with pTYB12-cdb3 expression vector were also transformed into HMS174 (DE3) for expression. The procedure was same as that for His-cdb3 before induction with IPTG. The culture medium was cooled to 25 °C before addition of IPTG to a final concentration of 0.1 mM. After additional 3 h incubation at 25 °C, the cells were collected by centrifugation at 6000g for 15 min. The harvested cells were frozen at )20 °C until use. Unless otherwise stated, all procedures were carried out at 0–4 °C. The cells were thawed and resuspended in 200 ml lysis buffer II (50 mM Tris–HCl, pH 8.0, 500 mM NaCl, and 200 lg/ml PMSF). The cells were incubated on ice for 15 min and then lysed by sonication, and the cellular debris was removed by centrifugation at 20,000g for 20 min. The supernatant was then loaded on a 20 ml
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(10 1.6 cm) column of chitin beads equilibrated with lysis buffer II. After washing the column with 200 ml lysis buffer II at the flow rate of 1 ml/min, the column was quickly flushed with 60 ml lysis buffer II containing 50 mM DTT at 20 °C and left for additional 40 h. The cleaved cdb3 was eluted with lysis buffer II and loaded on a Sephadex G-50 column (30 1.6 cm) equilibrated with 5 mM sodium phosphate buffer, pH 7.4. The desalted protein was collected and concentrated by Amicon Ultra-15 centrifugal filter at 4000g for 20 min. An equal volume of glycerol was added to make a final glycerol concentration of 50% followed by storage at )20 °C. Purification of cdb3 from the human erythrocytes Purification of cdb3 from the erythrocytes was performed according to Appell and Low [19]. Briefly, the fresh erythrocytes were washed with PBS (150 mM NaCl, 5 mM sodium phosphate buffer, pH 7.4) and then hemolyzed in 5 mM sodium phosphate buffer, pH 7.4, followed by centrifugation at 20,000g for 15 min. Following washing three times, the ghosts were incubated in 0.5 mM EDTA, pH 8.0, at 37 °C for 30 min to deplete spectrin and actin, and the ghosts were collected again by centrifugation at 20,000g for 15 min. The ankyrin, protein 4.1, and protein 6 were removed by incubating the pellets in 10 volumes of 0.17 M acetic acid for 20 min at 37 °C. The vesicles were collected by centrifugation at 20,000g for 15 min, washed in 0.1 M sodium phosphate buffer, pH 7.4, and suspended in 20 volumes of ice-cold 10 mM sodium phosphate buffer, pH 7.4. The cdb3 fragment was then cleaved from the vesicles by treatment of the vesicle suspension with 1 lg/ml a-chymotrypsin at 0 °C for 20 min. The digestion was terminated by addition of 200 lg/ml PMSF. The cdb3 was collected by centrifugation. The supernatant was collected and concentrated by Amicon Ultra-15 centrifugal filter at 4000g for 20 min. An equal volume of glycerol was added to make a final glycerol concentration of 50% followed by storage at )20 °C. SDS–PAGE and Western blot The protein samples (typically 10 ll) were mixed with 5 ll of 3 loading buffer [150 mM Tris–HCl (pH 6.8), 300 mM DTT, 6% (w/v) SDS, 0.06% (w/v) bromophenol blue, and 30% (v/v) glycerol], and boiled for 3 min prior to loading on a 10 or 12% Tris–HCl gel for electrophoresis using Bio-RadÕs Mini-PROTEIN 3 Cell. Proteins in the gel were visualized by staining with Coomassie G-250. The purity of stained proteins was determined by UVPÕs GelWorks gel analysis software and the protein concentration was determined by BCA method according to the protocol of BCA Protein Assay Reagent Kit (Pierce).
For Western blot analysis, the proteins were electrophoretically transferred onto 0.20 lm nitrocellulose transfer membrane (Schleicher & Schuell). The nitrocellulose was blocked by incubation in blocking buffer [150 mM NaCl, 50 mM Tris–HCl, pH 7.4, and 5% (w/v) low fat milk powder] for 1 h at 37 °C. Then, the membrane was incubated with anti-band 3 monoclonal antibody (Sigma) (1:5000) in the blocking buffer for 1 h at 37 °C followed by washing four times with washing buffer [150 mM NaCl, 50 mM Tris–HCl, pH 7.4, and 0.1% (v/v) Tween 20] for 10 min each wash. The membrane was then incubated with goat anti-mouse secondary antibody conjugated with HRP (1:5000) for 1 h at 37 °C. Also, the unbound HRP-conjugated secondary antibody was removed by washing with washing buffer four times for 10 min each wash. Finally, the membrane was incubated with working solution (SuperSignal WestPico Substrate) for 1 min and then pressed against an autoradiographic film. Determination of the molecular weight of recombinant proteins by mass spectrometry The purified recombinant proteins were desalted using HP 1100 LC system with column Zorbax 300SBC18. A linear gradient was formed by deionized water and 80% acetonitrile in 35 min. The eluate of peak fraction was collected and loaded on a mass spectrometer (PE API 165 LC/MS System) for measurement of the molecular weight. The pH-dependent property of the intrinsic fluorescence of recombinant cdb3 The measurement of intrinsic fluorescence of cdb3 was according to Low and co-workers [19,21]. Briefly, the purified recombinant cdb3 was dialyzed against different pH buffers containing 50 mM sodium phosphate, 50 mM sodium borate, 70 mM NaCl, and with or without 6 M urea. The protein concentration was determined by BCA method. The concentration of cdb3 in all measurement samples was 40 lg/ml. Fluorescence measurements were performed at 25 °C with a Hitachi M 850 spectrofluorometer. The wavelength of excitation was 290 nm and the fluorescence emission at 300–450 nm was detected. Expression of GST-Protein 4.2 C3 and GST The plasmid pGEX3X-Protein 4.2 C3 was a gift of Dr. J. Basu (Bose Institute, India). Protein 4.2 C3, which encompasses amino acid residues 187–260 of protein 4.2, is a critical domain for interaction of protein 4.2 with cdb3 [9]. The plasmid was transformed into E. coli HMS174 (DE3) and the expression was carried out in the presence of 0.1 mM IPTG at 25 °C for 3 h. The cells
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were collected, suspended in lysis buffer III (50 mM Tris–HCl, pH 8.0, 5 mM EDTA, 50 mM NaCl, 1% (v/v) Triton X-100, and 200 lg/ml PMSF), and sonicated on ice bath. The supernatant was collected by centrifugation at 20,000g for 20 min, loaded on a glutathione–Sepharose 4B column (5 0.7 cm), and washed with Tris–HCl buffer, pH 8.0, containing 1% (v/v) Triton X-100. Then, the protein was eluted with elution buffer (50 mM glutathione in Tris–HCl buffer, pH 8.0, and 1% (v/v) Triton X-100). Finally, the eluate was loaded on a Sephadex G50 column (30 1.6 cm) equilibrated with 5 mM sodium phosphate buffer, pH 7.4, to desalt. The desalted protein was collected and concentrated by Amicon Ultra-15 centrifugal filter at 4000g for 20 min. An equal volume of glycerol was added to make a final glycerol concentration of 50% followed by storage at )20 °C. The recombinant protein GST was expressed using pGEX-5X-1 vector and the same was purified as recombinant protein GST-Protein 4.2 C3. Pull-down assay The purified GST-Protein 4.2 C3 protein (10 lg) or control GST protein (8 lg) was loaded on the glutathione–Sepharose 4B resin (100 ll of 50% slurry) balanced with PBS containing 0.1% (v/v) Triton X-100. Hundred micrograms of the target protein [(N)His6-cdb3, (C)His6-cdb3, cleaved CBD-cdb3 or native cdb3 purified from the erythrocytes] was mixed with BSA (100 lg) and loaded on the resin (total volume 200 ll, 200 lg/ml PMSF). The mixture was loaded on Spin X Cups (Seize X Immunoprecipitation Kit, Pierce) by gently shaking overnight at 4 °C and centrifuged at 2000g for 30 s. After washing the resin four times with PBS containing 0.1% (v/v) Triton X-100, the proteins were eluted by SDS–PAGE loading buffer. The eluate was analyzed by SDS–PAGE followed with Western blot.
Results and discussion Cloning of the cdb3 to vectors pT470 and pTYB12 Both the pT7470 and pTYB12 vectors contain T7 polymerase promoter and can be induced by IPTG. For recombinant protein (N)His6-cdb3, the cdb3 gene was amplified with primers that introduced a BamHI site at its 50 end and a stop codon followed by a XhoI site at its 30 end. The first methionine residue encoded by the gene was not included because the vector pT7470 already contained it. It was verified by DNA sequencing that the expressed fusion protein (N)His6-cdb3 consists of 387 residues, compared with original cdb3 residues 1–379, due to additional eight residues (His–His–His–His–HisHis–Gly–Ser) inserted between the N-terminal first me-
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thionine residue and second glycine residue. For recombinant protein (C)His6-cdb3, the cdb3 gene was amplified with primers with a NdeI site at its 50 end and a XhoI site at its 30 end. It was verified by DNA sequencing that the expressed fusion protein (C)His6-cdb3 consists of 387 residues, compared with original cdb3 residues 1–379, due to additional eight residues (Leu–Glu–His–His–His–His–His–His) inserted in the C-terminal. For plasmid pTYB12, the cdb3 gene was amplified with primers that introduced a NdeI site at its 50 end and a stop codon followed by a XhoI site at its 30 end. The expressed protein has additional three residues (Ala–Gly–His) attached to the N-terminal after the affinity chitin-binding tag was self-cleaved, which was also verified by DNA sequencing. Expression and purification of cdb3 with hexahistidine tag To optimize the expression conditions, first, we tested the influence of temperature. It was observed that when induction time was 4 h, the expression of His6-cdb3 at 30 °C was higher than at 37 or 25 °C, and in all tested temperatures, most of the His6-cdb3 was in soluble form. Second, we tested the influence of IPTG concentration (0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, and 5 mM) and found that the yield was linear to IPTG below 1 mM concentration, but when IPTG concentration exceeded 1 mM, the yield did not increase significantly. Finally, we found that when induction time exceeded 4 h under the condition of 1 mM IPTG and 30 °C, the (N)His6cdb3 yield (about 30 mg from 1 L culture medium) did not increase, but (C)His6-cdb3 still increased up to about 6 h. So all further expression of His6-cdb3 was conducted using 1 mM IPTG at a growth temperature of 30 °C, the inducing time of (N)His6-cdb3 and (C)His6cdb3 was 4 and 6 h, respectively. We have tried to purify the His6-cdb3 protein by directly loading the lysate on the Ni–NTA Superflow resin, but there were always some contaminating proteins after elution (using this one-step method, the purity of His6-cdb3 was about 95% by SDS–PAGE analysis), no matter increasing the concentration of imidazole in the elution buffer, prolonging the wash time or using gradient elution. For increase in the purity of the recombinant protein, an anion exchanger step was added. The His6-cdb3 then was eluted from a DEAE– Sepharose CL-6B column at the NaCl concentration about 0.4 M. For desalinization, the method of dialysis was not desirable because it was time consuming and some of the proteins were easy to degrade during dialysis though the operation was performed at 4 °C. Therefore, we used gel filter instead. The final purity of His6-cdb3 was above 98% by SDS–PAGE assay. We have observed that after desalinization, the 43 kDa His6-cdb3 protein became unstable and easy to degrade to a 41 kDa fragment. So,
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it was immediately ultrafiltered and then an equal volume of glycerol was added to make a final glycerol concentration of 50% and stored at )20 °C to avoid degradation. Expression and purification of cdb3 with affinity chitinbinding tag We found that the majority of the expressed fusion protein CBD-cdb3 induced by IPTG was in the inclusion body by SDS–PAGE analysis. In an attempt to increase the yield of soluble and active CBD-cdb3, several experiments using different induction temperatures were carried out. When the temperature was decreased from 37 to 25 °C, the soluble fraction was increased, though not significantly. To choose the induction time it was found that 3 h was enough and longer induction did not result in improvement of soluble fraction. For impact of IPTG under condition of 25 °C and induction for 3 h, the final concentrations of IPTG from 0.02 to 5 mM were tested. The result showed that 0.1 mM IPTG was enough to give the highest expression of soluble CBDcdb3 (data not shown). The yield of soluble CBD-cdb3 was about 1.5 mg from 1 L culture medium, which was enough for further analysis of protein–protein interaction. So the optimization condition we chose was induction at 25 °C for 3 h with 0.1 mM IPTG. We used the inducible self-cleavage activity of the protein splicing element in CBD tag to separate the target-cleaved cdb3 protein (43 kDa) in the C-terminal region of the fusion protein from the chitin-binding affinity tag (58 kDa). The self-cleavage procedure does need not other expensive proteases and it happens just under high thiol (such as DTT, b-mercaptoethanol or cysteine) circumstances. After reaction with thiol reagent, the N-terminal is first cleaved and then the Cterminal region is cleaved [24,25]. After cleavage, the CBD fragment still bound with the chitin bead, the cleaved cdb3 protein was in the eluted cleavage buffer. During the self-cleavage testing, if the NaCl concentration was lower than 100 mM, most of the CBD-cdb3 was not spliced and was still bound to the chitin bead as 101 kDa fusion protein. Meanwhile, the protein should be exposed to room temperature for 40 h, which would increase the possibility of the degradation of recombinant cdb3. However, we found that when there were high salt (0.5 M NaCl) and high thiol (50 mM DTT), the protein was stable during purification. Because the purification depends on thiol-induced intein self-cleavage mechanism, the eluate after self-cleavage contained not only cleaved recombinant cdb3, but also a small peptide of 15 residues which had a molecular weight of about 1.6 kDa [25]. The result by use of Sephadex G-50 resin for further purification showed that CBD-cdb3 was efficiently separated from this small peptide and the high concentration salt in elution buffer.
Verification of purification by SDS–PAGE, Western blot, and mass spectrometry Figs. 1–3 show the typical SDS–PAGE and Western blot analysis for the purification of (N)His6-cdb3, (C)His6-cdb3, and CBD-cdb3, respectively. The results showed that the leakage expression can be neglected before IPTG induction, so it was not necessary to test the strain pLysS or pLysE. Lane 6 in Fig. 1 was the Western blot result of induced bacteria with band 3 antibody. A faint band about 31 kDa can been seen, which was the degraded cdb3 during induction. The degraded cdb3 can be efficiently separated from the fulllength recombinant cdb3 by DEAE anion exchanger. There was a minor leakage expression of (C)His6-cdb3 in unduced bacteria (Fig. 2, lane 7), but it did not interfere with the latter purification. There were some degraded fusion proteins when pTYB12-cdb3 plasmid was induced (Fig. 3, lane 10). But most of them were in the inclusion body and did not appear in the soluble CBD-cdb3 fraction (Fig. 3, lane 11). Tables 2–4 summarize the results of purification. The purity of stained proteins was determined by UVPÕs GelWorks gel analysis software and the concentration of proteins was determined by BCA method. The typical yields of (N)His6-cdb3, (C)His6-cdb3, and CBDcdb3 were about 10.6, 9.6, and 1.5 mg from 1 L culture medium, respectively. The calculated molecular weights of (N)His6-cdb3, (C)His6-cdb3, and the cdb3 cleaved from CBD-cdb3 are
Fig. 1. SDS–PAGE and Western blot analysis of (N)His6-cdb3. Lane 1, uninduced bacterial lysate; lane 2, induced bacterial lysate; lane 3, eluate from DEAE–Sepharose CL-6B anion exchanger; and lane 4, eluate from Ni–NTA resin purification. Lanes 5–8 represent the Western blot result of lanes 1–5 using anti-band 3 antibody.
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Table 2 Purification of recombinant (N)His6-cdb3 from an E. coli expression systema
Supernatant Anion exchanger Ni–NTA Gel filtration a
Total protein (mg)
(N) His6-cdb3 (mg)
Fold enrichment
Relative yield
86.5 20.6 13.0 10.8
27.7 19.0 12.7 10.6
1 2.9 3.0 3.1
1 0.69 0.46 0.38
From 1 L of E. coli culture medium.
Table 3 Purification of recombinant (C)His6-cdb3 from an E. coli expression systema
Fig. 2. SDS–PAGE and Western blot analysis of (C)His6-cdb3. Lane 1, NovagenÕs Perfect Protein markers; lane 2, uninduced bacterial lysate; lane 3, induced bacterial lysate; lane 4, eluate from DEAE– Sepharose CL-6B anion exchanger; and lane 5, eluate from Ni–NTA resin purification. Lanes 6–10 represent the Western blot result of lanes 1–5 using anti-band 3 antibody. S-Protein HRP conjugate in the Perfect Protein Western blot kits was used to detect the NovagenÕs Perfect Protein markers in Western blot analysis.
Supernatant Anion exchanger Ni–NTA Gel filtration a
Total protein (mg)
(C) His6-cdb3 (mg)
Fold enrichment
Relative yield
121 25.7 14.9 9.8
32.7 23.4 14.6 9.6
1 3.4 3.6 3.6
1 0.72 0.45 0.29
From 1 L of E. coli culture medium.
Table 4 Purification of recombinant CBD-cdb3 from an E. coli expression systema Total CBD-cdb3 protein (mg) (mg) Supernatant Chitin beads Gel filtration
117 2.13 1.57
Folder enrichment
2.52 (5.85)b 1 2.05 44.7 1.54 45.5
Relative yield 1 0.81 0.61
a
From 1 L of E. coli culture medium. The value was calculated according to the protein molecular weight before (101 kDa) and after (43 kDa) self-cleavage; the value in parentheses was the origin data gained from gel band density of 101 kDa CBD-cdb3 protein. b
on the measurement. They all accorded with the calculated molecular weights from the respective DNA sequences. Fig. 3. SDS–PAGE and Western blot analysis of CBD-cdb3. Lane 1, cdb3 purified from erythrocyte ghosts by digestion with a-chymotrypsin; lane 2, NovagenÕs Perfect Protein markers; lane 3, uninduced bacterial lysate; lane 4, induced bacterial lysate; lane 5, the proteins that bound with chitin beads after washing; and lane 6, eluate of selfcleaved proteins. Lanes 7–12 represent the Western blot result of lanes 1–6 using anti-band 3 antibody. S-Protein HRP conjugate in the Perfect Protein Western blot kits was used for detecting the NovagenÕs Perfect Protein markers in Western blot analysis.
43502.68, 43600.80, and 42,800.98 Da, respectively, by ExPASyÕs Peptide Mass Program. The two purified proteins were verified by mass spectrometry (Fig. 4, (C)His6-cdb3 was not tested). The molecular weights of (N)His6-cdb3 and the cdb3 cleaved from CBD-cdb3 were 43,501.0 Da, and 42,805.0 Da, respectively, based
The intrinsic fluorescence of the recombinant cdb3 One of the most important characters of the cdb3 protein is its ability to undergo a large and fully reversible pH-dependent conformational change [27]. The intrinsic fluorescence emission spectrum is one of the convenient ways to monitor this kind of structural change. There are four tryptophan residues in the cdb3 protein. In our experiment, the intrinsic fluorescence spectra were mainly contributed by these tryptophan residues. It is well known that both the fluorescence intensity and emission peak will be changed if the surrounding of the tryptophan residue changes. Especially, the fluorescence intensity will be decreased after tryptophan residue moves from hydrophilic
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Fig. 4. Verification of the recombinant cdb3 by mass spectrometry. (Left) (N)His6-cdb3 fusion protein and (right) the cdb3 cleaved from CBD-cdb3 fusion protein.
environment to hydrophobic environment. We have tested the recombinant proteins (N)His6-cdb3, (C)His6cdb3, and cleaved CBD-cdb3, found that the intrinsic fluorescence increased significantly when the pH increased, and meanwhile the emission peak moved to a longer wavelength. The shapes of the fluorescence spectrum among these three recombinant cdb3 and the
cdb3 purified from the human erythrocytes were similar to each other, especially at higher pH (see Fig. 5). When denatured in 6 M urea, the fluorescence emission peaks moved to about 350 nm and the pH dependence of the intrinsic fluorescence disappeared (data not shown). The character of the pH dependence of the intrinsic fluorescence demonstrated that the recombinant proteins were structural active. Pull-down assay Protein 4.2 is a peripheral membrane protein that presents in 200,000 copies per erythrocyte and comprises approximately 5% of the total membrane proteins of the erythrocyte. It is commonly believed that the interaction between band 3, ankyrin, and protein 4.2 is one of the most important linkages between the skeleton network and the plasma membrane in the erythrocytes, although the information on the binding site of protein 4.2 on cdb3 is limited [22]. C3 is a protein 4.2 fragment of residues 187–260, a domain critical for the protein 4.2–cdb3 interaction [9]. Using pull-down assay, the ability of interaction between GST-Protein 4.2 C3 and cdb3 was studied
Fig. 5. pH dependence of the intrinsic fluorescence of recombinant cdb3. The relative intensity of the fluorescence emission from 300 to 450 nm was plotted (the excitation wavelength was 290 nm). The emission spectra of the recombinant proteins were obtained at pH values of 10.0, 9.0, 8.0, 7.0, and 6.0 (upper to lower). (A) The cdb3 purified from the human erythrocytes; (B) the cdb3 cleaved from CBDcdb3 fusion protein; (C) (N)His6-cdb3 fusion protein; and (D) (C)His6-cdb3 fusion protein.
Fig. 6. Association of recombinant cdb3 with protein 4.2. The purified GST-Protein 4.2 C3 (lanes 3, 5, 7, and 9) or control GST protein (lanes 2, 4, 6, and 8) was loaded on the glutathione–Sepharose 4B resin balanced with PBS containing 0.1% (v/v) Triton X-100. The target protein, cdb3 purified from erythrocyte (lanes 2 and 3), (N)His6-cdb3 (lanes 4 and 5), (C)His6-cdb3 (lanes 6 and 7), or cleaved CBD-cdb3 (lanes 8 and 9), was loaded on the resin by gently shaking overnight at 4 °C. After washing, the proteins eluted by SDS–PAGE loading buffer were analyzed by SDS–PAGE followed with Western blot using antiband 3 antibody. Lane 1, NovagenÕs Perfect Protein markers.
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here. Both (N)His6-cdb3, (C)His6-cdb3, and CBD-cdb3 can react with protein 4.2 C3 fragment (Fig. 6). Using purified GST as a control, it was observed that GST could not pull (N)His6-cdb3, (C)His6-cdb3 or CBD-cdb3 down. The pull-down assay to some extent verified that the recombinant proteins were functionally active. In summary, we have developed novel methods to express cdb3 with tags for further research, especially in protein–protein interaction and crystallography study. All the three recombinant cdb3 were both structurally and functionally similar to the native cdb3. The yields of cdb3 with hexahistidine tag in N- or C-terminal were similar and much higher than CBD-cdb3. The purification procedures were timesaving and suitable for getting enough homogeneous proteins with high purity for further research. Meanwhile, cleaved CBD-cdb3 was much more stable than His6-cdb3 after long time preservation. Acknowledgments The work was supported by Grant 2001CB5102 from the National Basic Research Priorities Program of China and the grants (30070205 and 30370380) from the National Natural Science Foundation of China. The authors thanks Yi Zhang at the Research Center of Biotechnology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, for advice on construction of the vector pT7470. References [1] P.S. Low, Structure and function of the cytoplasmic domain of band 3: center of erythrocyte membrane-peripheral protein interactions, Biochim. Biophys. Acta 864 (1986) 145–167. [2] S.E. Lux, K.M. John, R.R. Kopito, H.F. Lodish, Cloning and characterization of band 3, the human erythrocyte anionexchange protein (AE1), Proc. Natl. Acad. Sci. USA 86 (1989) 9089–9093. [3] C.C. Wang, R. Moriyama, C.R. Lombardo, P.S. Low, Partial characterization of the cytoplasmic domain of human kidney band 3, J. Biol. Chem. 270 (1995) 17892–17897. [4] V. Bennett, P.J. Stenbuck, Association between ankyrin and the cytoplasmic domain of band 3 isolated from the human erythrocyte membrane, J. Biol. Chem. 255 (1980) 6424–6432. [5] C.R. Lombardo, B.M. Willardson, P.S. Low, Localization of the protein 4.1-binding site on the cytoplasmic domain of erythrocyte membrane band 3, J. Biol. Chem. 267 (1992) 9540–9546. [6] J.C. Pinder, A. Chung, M.E. Reid, W.B. Gratzer, Membrane attachment sites for the membrane cytoskeletal protein 4.1 of the red blood cell, Blood 82 (1993) 3482–3488. [7] C. Korsgren, C.M. Cohen, Associations of human erythrocyte band 4.2. Binding to ankyrin and to the cytoplasmic domain of band 3, J. Biol. Chem. 263 (1988) 10212–10218. [8] A.C. Rybicki, S. Musto, R.S. Schwartz, Identification of a band-3 binding site near the N-terminus of erythrocyte membrane protein 4.2, Biochem. J. 309 (Pt 2) (1995) 677–681. [9] R. Bhattacharyya, A.K. Das, P.K. Moitra, B. Pal, I. Mandal, J. Basu, Mapping of a palmitoylatable band 3-binding domain of human erythrocyte membrane protein 4.2, Biochem. J. 340 (Pt 2) (1999) 505–512.
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