- Email: [email protected]
CBM21 starch-binding domain: A new purification tag for recombinant protein engineering
Protein Expression and Purification 65 (2009) 261–266
Contents lists available at ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
CBM21 starch-binding domain: A new purification tag for recombinant protein engineering Shu-Chuan Lin a, I-Ping Lin a, Wei-I Chou b, Chen-An Hsieh a, Shi-Hwei Liu b, Rong-Yuan Huang a, Chia-Chin Sheu b, Margaret Dah-Tsyr Chang a,* a b
Institute of Molecular and Cellular Biology, Department of Life Science, National Tsing Hua University, Hsinchu 300, Taiwan, ROC Simpson Biotech Co., Ltd., Taoyuan County 333, Taiwan, ROC
a r t i c l e
i n f o
Article history: Received 10 December 2008 and in revised form 12 January 2009 Available online 23 January 2009 Keywords: Rhizopus oryzae Glucoamylase Starch-binding domain Enhanced green fluorescent protein Protein purification Carbohydrate-binding module
a b s t r a c t The use of protein fusion tag technology simplifies and facilitates purification of recombinant proteins. In this article, we have found that the starch-binding domain derived from Rhizopus oryzae glucoamylase (RoSBD), a member of carbohydrate-binding module family 21 (CBM21) with raw starch-binding activity, is favorable to be applied as an affinity tag for fusion protein engineering and purification in Escherichia coli and Pichia pastoris systems. To determine suitable spatial arrangement of RoSBD as a fusion handle, enhanced green fluorescent protein (eGFP) was fused to either the N- or C-terminus of the SBD, expressed by E. coli, and purified for yield assessment and functional analysis. Binding assays showed that the ligand-binding capacity was fully retained when the RoSBD was engineered at either the N-terminal or the C-terminal end. Similar results have been obtained with the RoSBD-conjugated phytase secreted by P. pastoris. The effective adsorption onto raw starch and low cost of starch make RoSBD practically applicable in terms of development of a new affinity fusion tag for recombinant protein engineering in an economic manner. Ó 2009 Elsevier Inc. All rights reserved.
In nature, many amylolytic enzymes, such as a-amylase, b-amylase, and glucoamylase (GA),1 possess specific raw starchbinding ability through their carbohydrate-binding modules (CBMs). CBMs are defined protein domains capable of binding polysaccharide moieties. To date within 53 CBM families, eight representative members are characterized to possess starch-binding activities and thus named as starch-binding domains (SBDs). Among which the N-terminal SBD of Rhizopus oryzae glucoamylase (RoSBD) belongs to CBM family 21 and shares a relatively low level of similarity with the SBDs derived from the other starch-processing enzymes. RoSBD is a functional ligand-binding domain and has been demonstrated to be effectively adsorbed onto raw starch and other soluble oligosaccharides [1,2]. It has a molecular weight of 12 kDa and is classified as a member of Type B glycan chain-binding CBM [3]. The location and orientation of the aromatic residues in the binding sites of Type B CBMs are discovered to serve as the major factors in determining ligand affinity and specificity [4,5]. We have previously demonstrated that RoSBD possesses two ligand-binding sites, where hydrophobic interactions between the sugar rings and six aromatic
* Corresponding author. Fax: +886 3 5715934. E-mail address: [email protected] (M.D. Chang). 1 Abbreviations used: GA, glucoamylase; CBM, carbohydrate-binding module; SBD, starch-binding domain; MBP, maltose-binding protein; eGFP, enhanced green fluorescent protein; CBD, carbohydrate-binding domain; WSSV, white spot syndrome virus; HIV, human immunodeficiency virus. 1046-5928/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2009.01.008
residues on RoSBD surface play crucial roles. Specifically the residues Tyr32, Phe58, and Tyr67 constitute the binding site I, whereas the residues Trp47, Tyr83, and Tyr94 form the binding site II [2]. A large repertoire of protein purification tags is now available [6], such as polyhistidine (Poly-His), maltose-binding protein (MBP) and glutathione S-transferase (GST) [7–9]. The major obstacle that hinders these tags to be developed into large-scale bioprocessing technique is the cost of the affinity resin and other required reagents. In this paper, we describe the procedures for purifying SBD-fusion proteins based on raw starch adsorption–elution method. Raw starch is a suitable material for affinity separation of proteins due to its abundance, stability, nontoxicity, and easy recovery [10,11], hence large-scale manufacture process using raw starch seems very feasible. Our RoSBD possesses a high binding affinity towards raw starch [1]. Starch is an abundant polysaccharide composed of two types of D-glucose polymers: amylose and amylopectin. The former is composed of mostly linear a-1,4-linked glucose residues, whereas the latter is the highly branched component of starch containing 5–6% of a-1,6-linkages [12]. We use two different heterologous expression systems, Escherichia coli and Pichia pastoris to demonstrate the performance of the recovery system. For E. coli expression system, enhanced green fluorescent protein (eGFP) [13] was used as the target protein because its fluorescence could be observed only when it was correctly folded [14]. For P. pastoris expression system, phytase [15,16], an enzyme in poultry feed to lower phosphorus levels in poultry litter [17,18], was se-
262
S.-C. Lin et al. / Protein Expression and Purification 65 (2009) 261–266
lected as a candidate for secretory protein expression and purification. Materials and methods Strains, media, and plasmids Escherichia coli TOP10F’ (Invitrogen) was used for plasmid manipulation, while the E. coli strain BL21-CodonPlusÒ (DE3) (Stratagene) and the P. pastoris strain GS115 were used for protein expression. E. coli cells were grown in Luria–Bertani (LB) medium (1% tryptone, 0.5% yeast extract, and 0.5% sodium chloride) containing 50 lg/ml ampicillin at 37 °C. P. pastoris cells were grown in yeast/peptone/dextrose medium (YPD; 1% yeast extract, 2% peptone, and 2% dextrose) or buffered glycerol-complex (BMGY; 1% yeast extract, 2% peptone, 1% glycerol, 1.34% YNB, 4 10 5%, 100 mM potassium phosphate, pH 6.0) medium at 30 °C. The plasmid vectors used in this work were as follows: pGEMÒ-T Easy vector (Promega), pET23a (+) (Novagen), pPIC9 (Invitrogen), and pPICZaA (Invitrogen). Construction of the recombinant plasmids The fragments encoding RoSBD, eGFP, and phytase were individually amplified by PCR following standard protocols (Table 1). Amplified products were analyzed on 1% agarose gel, purified and ligated to pGEM-T vector, and verified by DNA sequencing. These fragments were then individually ligated into E. coli or P. pastoris expression vectors. Expression of fusion protein in E. coli The pET expression vector was transformed into BL21-CodonPlusÒ (DE3) using heat shock method. Protein expression was induced by addition of 0.5 mM IPTG to the cultures and incubation at 20 °C for 16 h. Cells were harvested by centrifugation, washed and then disrupted by sonication. Sonicated suspensions were centrifuged at 10,000g for 30 min at 4 °C and the supernatants were sterile filtered (0.2 lm). Expression of fusion protein in P. pastoris Transformation of Pichia was performed by lithium chloride method according to the manufacturer’s protocol (Invitrogen). Ten micrograms of the recombinant plasmid, pPICZaA/sbd-phytase or pPIC9/phytase-sbd was linearized using restriction enzyme PmeI and electroporated into P. pastoris GS115. Positive colonies
were selected in YPD plates containing 100 lg/mL Zeocine and sequentially induced for recombinant protein expression by culturing them in BMGY, followed by buffered methanol-complex medium (BMMY) containing 0.75% methanol. After 72 h, culture supernatants were collected by centrifugation at 3000g for 30 min at 4 °C. Purification of SBD and SBD-fusion proteins RoSBD was purified using prepacked HiTrapTM SP column [19]. All SBD-fusion proteins were purified by affinity chromatography using an amylose resin column. A 2-mL bed of amylose resin (New England Biolabs) was poured into a 1.5 10 cm column, equilibrated in buffer (50 mM NaOAc, pH 5.5), and the clarified cell lysate containing the SBD-fusion protein was loaded onto the column. The column was then washed with binding buffer (50 mM NaOAc, pH 5.5) and the adsorbed proteins were eluted with elution buffer (10 mM glycine/NaOH, pH 11). In addition, two purification methods were developed using raw starch in this study. One was stirring method, referred to Fang’s method [20]. Fifty milligrams corn starch (Sigma–Aldrich, EC 232-679-6) were washed three times in elution buffer and binding buffer as described above. Prewashed starch was suspended in 1 mL protein solution with slow rotation on a platform shaker at 25 °C for 3 h. The starch pellet with adsorbed proteins was washed with 3 mL of binding buffer, and then eluted by 1.5 mL elution buffer. The other was columnbased method developed by modifying a method proposed by Lin [21]. The corn starch (200 mg) was washed as described for the stirring method and packed into a 5-mL column in which a filter paper was placed at the bottom. After the protein solution (3 mL) was loaded onto the column, the column was washed with 6 mL binding buffer and the adsorbed proteins were eluted with 3 mL elution buffer. The protein purity was determined by 12% or 15% SDS–PAGE and protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent kit (Pierce), with BSA as the reference standard. Association rate assay of SBD The purified SBD in the concentration range from 15.6 to 27.2 lM was mixed with prewashed corn starch (1 mg/mL) in a final volume of 100 lL and incubated at 25 °C with gentle stir for 5 h. The binding was terminated at different time intervals by sedimentation of the starch. After centrifugation at 16,000g for 10 min at 4 °C, the protein concentration in the supernatant (unbound protein) was determined by the BCA assay, and the amount of bound protein was calculated from the difference between the ini-
Table 1 Plasmids generated and used in this study. Name of the plasmids
Description
Ref.
pET23a/sbd pET23a/egfp–sbd
The sbd gene was cloned into the NdeI/XhoI-restricted pET23a vector. The cloned egfp and sbd were excised with EcoRI/HindIII and HindIII/XhoI and inserted together into the EcoRI and XhoI sites of pET23a vector. The cloned sbd and egfp were excised with NdeI/KpnI and KpnI/XhoI and inserted together into the NdeI and XhoI sites of pET23a vector. The cloned sbd-l58 and egfp were excised with NdeI/KpnI and KpnI/XhoI and inserted together into the NdeI and XhoI sites of pET23a vector. The cloned sbd-l38 and egfp were excised with NdeI/KpnI and KpnI/XhoI and inserted together into the NdeI and XhoI sites of pET23a vector. The cloned sbd-l8 and egfp were excised with NdeI/KpnI and KpnI/XhoI and inserted together into the NdeI and XhoI sites of pET23a vector. The gene fragment sbd-phytase was generated by two-step PCR and the amplified fragment was digested using EcoRI and inserted into the Pichia expression vector pPICZaA. The gene fragment phytase-sbd was generated by two-step PCR and the amplified fragment was digested using KpnI/EcoRI and inserted into the Pichia expression vector pPIC9.
[2,3,14] This study
pET23a/sbd–egfp pET23a/sbd-l58– egfp pET23a/sbd-l38– egfp pET23a/sbd-l8– egfp pPICZaA/sbdphytase pPIC9/phytase-sbd
This study This study This study This study This study This study
S.-C. Lin et al. / Protein Expression and Purification 65 (2009) 261–266
tial and unbound protein concentrations. The bound protein at equilibrium expressed as micromole of protein per gram of starch was a linear function of the free (unbound) protein in the range of protein concentrations assayed. Saturation binding assay The purified SBD or SBD-fusion protein at a concentration ranging from 10 to 120 lM was mixed with 1 mg of prewashed corn starch in 50 mM NaOAc, pH 5.5 and incubated at 25 °C for 3 h. After centrifugation at 16,000g for 10 min at 4 °C, the unbound protein was determined by BCA assay. The amount of adsorbed protein was calculated from the difference between the initial and unbound protein concentrations. Kinetic parameters were determined from the binding isotherms with non-linear regression (GraphPad Prism 4). Effects of pH on binding ability The purified protein with a concentration of 20 lM (0.25 mg/ mL) was added to 100 lL of prewashed corn starch (1 mg/mL) in buffer solution with different pH values and incubated at 25 °C for 1 h. The buffers used were 50 mM glycine/HCl (pH 2–3), sodium acetate/acetic acid (pH 4–5.5), Na2HPO4/NaH2PO4 (pH 6–7), Tris/ HCl (pH 8), and glycine/NaOH (pH 9–11). The free fusion protein concentration of the supernatant before and after the binding was determined by BCA assay. The relative binding ability of the fusion protein assayed at pH 5.5 was normalized as 100%.
263
Effects of starch on SBD adsorption One hundred milligrams of raw starch from various sources were prewashed and suspended in 1 mL binding buffer (50 mM NaOAc, pH 5.5). Thirty microliters (3 mg) of starch solution were added to 90 lL of protein solution (final concentration of 1 mg/mL), and the suspension was incubated at 25 °C with gentle stirring for 2 h. After incubation, the samples were centrifuged at 16,000g for 10 min at 4 °C, the unbound protein concentration of the supernatant was determined and the adsorption percentage was calculated. Results and discussion Characteristic properties of RoSBD The RoSBD structure revealed eight b-strands arranged essentially in an antiparallel fashion, forming an open-sided b-barrel organized into two b-sheets (Fig. 1A) [2,12]. As illustrated in Fig. 1A, the key aromatic residues in the ligand-binding sites I and II were indicated. The pH effects on the binding of RoSBD to corn starch indicated that the protein was able to tightly bind to raw starch at a pH range from 4.0 to 7.0. Such ligand binding of RoSBD appeared to be pH dependent, as the maximal binding was gained at pH 4.0 and pH 5.0, whereas the binding was significantly weakened at the pH values below 4.0 and above 8.0 (Fig. 1B). In order to determine the time needed to reach equilibrium for binding to starch, a time-course experiment was performed. In the initial period (up to 50 min), the adsorption exhibited a linear phase and the
Fig. 1. Characterization of RoSBD. (A) The structure of apo RoSBD (PDB code 2VQ4) with eight b-strands forming two b-sheets and a distorted barrel. (B) pH effect on the adsorption of RoSBD to corn starch. The binding ability of RoSBD at varying pH was measured as described in Materials and methods. (C) Time course of adsorption of RoSBD to corn starch. Purified protein at different concentrations (N, 15.6; j, 22.5, and d, 27.2 lM) was added to 0.1 mg of prewashed granular corn starch and incubated at 25 °C with gentle stirring for 5 h. (D) Adsorption isotherm of RoSBD binding to corn starch at pH 5.5 and 25 °C.
264
S.-C. Lin et al. / Protein Expression and Purification 65 (2009) 261–266
maximal binding was achieved after 2 h (Fig. 1C). Furthermore, the adsorption isotherm of RoSBD resulted in a Bmax of 45.5 ± 1.4 lmol/ g and Kd of 6.9 ± 0.7 lM (Fig. 1D). Purification of RoSBD by amylose affinity chromatography The adsorption of RoSBD to raw starch was strongly dependent on the pH, suggesting that electrostatic interactions play an important role in the adsorption process. Hence the recombinant RoSBD was affinity purified by one-step chromatography on cross-linked amylose (Fig. 2A), where the binding and elution steps were performed in buffers with pH values of 5.5 (50 mM NaOAc) and 11.0 (10 mM glycine/NaOH), respectively. The adsorption capacity of amylose resin was greater than 50 mg/mL, and the adsorbed SBD was eluted with the basic buffer to give high purity SBD exceeding 97% with this single-step purification scheme. An average yield of 30 mg purified RoSBD could be obtained from 1 L E. coli culture. RoSBD as a purification handle in E. coli expression system In order to evaluate the application of the RoSBD as a purification handle, recombinant proteins consisting SBD fused to eGFP were expressed in E. coli system. Both N- and C-terminal fusions could be produced and readily purified by the procedure as described above. Similar to the case of RoSBD, the 39 kDa recombinant SBD-fusion proteins with high purities exceeding 90% could be obtained (Fig. 2B). The system has a high capacity to purify recombinant fusion proteins in E. coli because the background of endogenous protein interacting with starch is quite low. These results confirmed that RoSBD could confer starch-binding activity in both N- and C-terminal fusions.
Fig. 3. Effects of various starches on the adsorption of SBD and SBD-fusion proteins. Three milligrams of starch was used for adsorbing the target protein at a concentration of 1 mg/mL in a total volume of 120 lL. After incubation at 25 °C for 2 h, the sample was centrifuged and the adsorption percentage was determined. Each data point represents the average of three independent measurements, and error bars represent SD.
Purified SBD–eGFP and eGFP–SBD were subsequently used to evaluate the application of native starch as an affinity adsorbent. To investigate the effects of starch from a variety of natural sources on the purification, each native starch was prepared by washing with 10 mM glycine/NaOH (pH 11) and 50 mM NaOAc (pH 5.5). The fusion protein was separately added to the washed starch (3 mg) in a total volume of 120 lL with initial protein concentration of 1 mg/mL, and the mixture was stirred at 25 °C for 2 h. In comparison with the adsorption percentage of SBD, SBD–eGFP, and eGFP–SBD with different kinds of starch, Fig. 3 revealed that the adsorption efficiency of corn, rice, sweet potato, tapioca, and wheat starch was greater than 90%, while that of the other three starches from mung bean, potato and soybean was less than 60%. Although corn, rice, sweet potato, tapioca, and wheat starch had comparable adsorption capacity, corn starch was chosen as the adsorbent for the following studies due to its lower cost. In addition, the SBD and the targeted eGFP fusion proteins showed similar properties in terms of starch binding. Purification of SBD-tagged fusion proteins by corn starch
Fig. 2. Purification of SBD using amylose resin. (A) E. coli cells from 200 mL of culture were collected by centrifugation. After lysis, cells were centrifuged to remove cell debris, and then loaded onto the amylose resin pre-equilibrated with binding buffer 50 mM NaOAc, pH 5.5. After wash with 30 mL equilibration buffer, 10 mM NaOAc, pH 11.0 was added to elute SBD and 10 mL fractions were collected. Lanes represent the column load (L), flow through (F), and wash (W), followed by four lanes of eluate (E). Samples (20 lL) were separated by 15% SDS–PAGE and stained with Coomassie blue. (B) SDS–PAGE (15%) analysis of purified SBD and SBDfusion proteins. SBD, SBD–eGFP, and eGFP–SBD were expressed in E. coli and the fusion proteins SBD-Phytase and Phytase-SBD were expressed and purified from the P. pastoris culture medium. Lanes 1, SBD; 2, SBD–eGFP; 3, eGFP–SBD; 4, SBDPhytase; and 5, Phytase-SBD.
Together with the application of natural starch, corn starch with the required properties was selected as a model to study its effectiveness. Here stirring and column-based methods were developed for protein purification. Three different fusion proteins with interdomain linkers of 8, 38, and 58 amino acid residues, named SBDL8–eGFP, SBD-L38–eGFP, and SBD-L58–eGFP, respectively, were tested. The characteristic starch-binding properties of SBD-L8– eGFP were quite similar to that of RoSBD (Supplementary Fig. 1). The affinity of SBD-L8–eGFP to corn starch was 2.7 ± 0.2 lM and the Bmax value was determined as 77.6 ± 1.5 lmol/g. Table 2 summarized the data from those SBD-fusion proteins with regard to the purification efficiency in terms of adsorption and recovery rate. The adsorption (%) is defined as [(original protein concentration supernatant protein concentration)/original protein concentration] 100. The recovery rate (%) is defined as (eluted protein
S.-C. Lin et al. / Protein Expression and Purification 65 (2009) 261–266 Table 2 Purification methods for recombinant SBD-fusion proteins. Protein samples
Adsorption (%)a
Recovery rate (%)b
Stirring-based method SBD-L58–eGFP SBD-L38–eGFP SBD-L8–eGFP
90.3 ± 1.2 94.6 ± 2.4 96.6 ± 6.7
50.0 ± 5.7 55.4 ± 11.9 44.9 ± 9.9
Column-based method SBD-L58–eGFP SBD-L38–eGFP SBD-L8–eGFP
84.7 ± 8.3 84.5 ± 12.0 82.0 ± 5.4
83.6 ± 9.2 86.0 ± 2.5 87.1 ± 4.1
The data are expressed as average value ± SD of three independent trials. a The adsorption (%) is defined as [(original protein concentration supernatant protein concentration)/original protein concentration] 100. b The recovery rate (%) is defined as (eluted protein concentration/adsorbed protein concentration) 100.
concentration/adsorbed protein concentration) 100. As an illustration, stirring method (Fig. 4A) for the preparation of adsorbent had high adsorption efficiency (90–97%), but the recovery rate was relatively low (50–55%). Alternatively, the column-based method (Fig. 4B) gave relatively lower adsorption efficiency (82– 85%), but the average recovery rate was 86%, much better than that of the stirring-based method. These results suggest that it is feasible to use corn starch as an adsorbent for the recovery and purification of recombinant fusion proteins. In addition, among the three SBD-fusion proteins with linker variants used in this study, insertion of the linker region between RoSBD and eGFP did not affect the starch-binding affinity. In comparison, although the starch adsorption rate was promoted in the stirring method, the recovery yield was significantly improved in the column-based one. Moreover, as the sample was forced to pass through a simple column packed with starch, rapid recovery of the target protein was achieved. However, the conse-
Fig. 4. Purification of SBD-fusion proteins using raw starch. The purification was carried out by (A) stirring and (B) column-based methods. Three SBD–eGFP fusion proteins with L8, L38, and L58 linkers containing 8, 38, and 58 amino acid residues were, respectively, indicated, lanes represent the size marker (M), column load (L), flow through (F), wash (W), and eluate (E). An equal volume (12 lL) of each sample was loaded onto 12% SDS–PAGE.
265
quence for lower adsorption efficiency in the system was possibly correlated with short contact time. The result indicated that the column-based method was more effective, and it could be further developed into a commercial purification technique due to the convenience, efficiency and economy. RoSBD as a purification tag in P. pastoris expression system Since E. coli is only one of several popular recombinant expression hosts, the practical application of SBD tag in P. pastoris expression system for production of secretory proteins was evaluated as well. Here E. coli phytase capable of degrading phytic acid was selected as the target protein. The column-based method was used to assess the ability of RoSBD for purification of secreted recombinant phytase (SBD-Phytase and Phytase-SBD) from the bulk yeast culture media. The absorption efficiency of these recombinant proteins (61–64%) was found to be lower than that of E. coli system (90–97%), presumably due to short contact time passing through the column or competition of oligosaccharides in the culture medium. In addition, 30–40% of the SBD-tagged proteins present in the culture media could be retrieved in the elution fraction. The purity of both SBD-Phytase and Phytase-SBD was higher than 90% according to SDS–PAGE analysis (Fig. 2B). Conclusion Affinity tags are important for facilitating protein purification [22]. Rapid and low-cost production of recombinant proteins is necessary for generation of industrially relevant proteins. Fusion protein technology has been enthusiastically pursued since the advent of genetic engineering. To add a molecular handle to a recombinant protein is an attractive way to simplify the purification scheme [23]. Many polysaccharide hydrolyzing enzymes display a carbohydrate-binding domain (CBD), and a few of them have been extensively studied for recombinant protein purification. For example, E. coli MBP is a commonly used tag for streamlined purification [23–25], but the relatively large size of MBP may preclude the structure or function of target protein. Moreover, the chitin-binding domain from Bacillus circulans was applied as the affinity tag to purify Staphylococcus simulans lysostaphin [26], but it required a high salt concentration (higher than 500 mM) or the use of non-ionic detergents to optimize the purity of the product, so this harsh condition might reduce its application potentiality. In addition, many CBDs with the size ranging from 4 to 20 kDa are currently available for purification purpose [22]. A major drawback is the stringent elution condition, which may compromise purification and further use of the tagged proteins. CBMs are now grouped into 53 families according to ligand specificity and 8 CBM family members are characterized to possess starch-binding activities. Based on the characteristics of starchbinding CBMs, starch and its derivatives have been used for the elution of a-amylase, b-amylase, glucoamylase, isoamylase, and cyclodextrin glucantransferase from the adsorbents [27]. Here the RoSBD, the first CBM21 member being structurally and functionally characterized, used in this study has relatively small size of 12 kDa and binds to starch over the pH range from 4.0 to 7.0. It can be used as either N- or C-terminal fusion partners for efficient purification. Moreover, we have also applied this system to purify two viral proteins at laboratory scale. For example, the envelope viral protein VP28 of white spot syndrome virus (VP28/WSSV) and the envelope glycoprotein protein GP41 of human immunodeficiency virus (GP41/HIV) were separately engineered with N-terminal SBD-fusion head. As expected, both proteins were successfully expressed and purified (Supplementary Fig. 2). There-
266
S.-C. Lin et al. / Protein Expression and Purification 65 (2009) 261–266
fore, a number of tested results verified that the purification method developed in this study is simple and efficient. In some applications, it may not be necessary to remove the RoSBD tag from the fusion proteins. For instance, phytase is a feed supplement to the diet of domesticated animals; using starch adsorption to recover the fusion proteins takes advantages of direct product collection, which needs no further separation. As a result, the properties of starch make it a better value for particular application. In summary, we have provided direct evidence showing that RoSBD can be attached to different proteins to facilitate purification on starch. The fusion proteins produced by E. coli and P. pastoris cells could be recovered with high purity by adsorption–elution procedure on raw starch. The RoSBD is thus demonstrated to offer a convenient, economical and easy solution to manifest recombinant protein engineering and purification. Acknowledgments This work is supported by Simpson Biotech Co., Ltd., Taiwan, ROC; National Science Council, ROC, Grant 97-2622-B-007-001; and Council of Agriculture and the National Science and Technology Program for Agricultural Biotechnology, Grant 97AS-1.2.1.ST-a5 to M. D.-T. Chang. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.pep.2009.01.008. References [1] W.I. Chou, T.W. Pai, S.H. Liu, B.K. Hsiung, M.D. Chang, The family 21 carbohydrate-binding module of glucoamylase from Rhizopus oryzae consists of two sites playing distinct roles in ligand binding, Biochem. J. 396 (2006) 469–477. [2] J.Y. Tung, M.D. Chang, W.I. Chou, Y.Y. Liu, Y.H. Yeh, F.Y. Chang, S.C. Lin, Z.L. Qiu, Y.J. Sun, Crystal structures of the starch-binding domain from Rhizopus oryzae glucoamylase reveal a polysaccharide-binding path, Biochem. J. 416 (2008) 27–36. [3] A.B. Boraston, D.N. Bolam, H.J. Gilbert, G.J. Davies, Carbohydrate-binding modules: fine-tuning polysaccharide recognition, Biochem. J. 382 (2004) 769– 781. [4] L. Guan, Y. Hu, H.R. Kaback, Aromatic stacking in the sugar binding site of the lactose permease, Biochemistry 42 (2003) 1377–1382. [5] T. Ponyi, L. Szabo, T. Nagy, L. Orosz, P.J. Simpson, M.P. Williamson, H.J. Gilbert, Trp22, Trp24, and Tyr8 play a pivotal role in the binding of the family 10 cellulose-binding module from Pseudomonas xylanase A to insoluble ligands, Biochemistry 39 (2000) 985–991. [6] M. Hedhammar, S. Hober, Z(basic)—a novel purification tag for efficient protein recovery, J. Chromatogr. A 1161 (2007) 22–28.
[7] D.B. Smith, K.S. Johnson, Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase, Gene 67 (1988) 31–40. [8] K. Terpe, Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems, Appl. Microbiol. Biotechnol. 60 (2003) 523–533. [9] M.B. Murphy, S.A. Doyle, High-throughput purification of hexahistidine-tagged proteins expressed in E. coli, Methods Mol. Biol. 310 (2005) 123–130. [10] L.J. Chen, C. Ford, A. Kusnadi, Z.L. Nikolov, Improved adsorption to starch of a beta-galactosidase fusion protein containing the starch-binding domain from Aspergillus glucoamylase, Biotechnol. Prog. 7 (1991) 225–229. [11] L.J. Chen, C. Ford, Z. Nikolov, Adsorption to starch of a beta-galactosidase fusion protein containing the starch-binding region of Aspergillus glucoamylase, Gene 99 (1991) 121–126. [12] Y.N. Liu, Y.T. Lai, W.I. Chou, M.D. Chang, P.C. Lyu, Solution structure of family 21 carbohydrate-binding module from Rhizopus oryzae glucoamylase, Biochem. J. 403 (2007) 21–30. [13] B.P. Cormack, R.H. Valdivia, S. Falkow, FACS-optimized mutants of the green fluorescent protein (GFP), Gene 173 (1996) 33–38. [14] N.J. Caron, Y. Torrente, G. Camirand, M. Bujold, P. Chapdelaine, K. Leriche, N. Bresolin, J.P. Tremblay, Intracellular delivery of a Tat–eGFP fusion protein into muscle cells, Mol. Ther. 3 (2001) 310–318. [15] C.C. Chen, P.H. Wu, C.T. Huang, K.J. Cheng, A Pichia pastoris fermentation strategy for enhancing the heterologous expression of an Escherichia coli phytase, Enzyme Microb. Technol. 35 (2004) 315–320. [16] H. Luo, H. Huang, P. Yang, Y. Wang, T. Yuan, N. Wu, B. Yao, Y. Fan, A novel phytase appA from Citrobacter amalonaticus CGMCC 1696: gene cloning and overexpression in Pichia pastoris, Curr. Microbiol. 55 (2007) 185–192. [17] N. Liu, G.H. Liu, F.D. Li, J.S. Sands, S. Zhang, A.J. Zheng, Y.J. Ru, Efficacy of phytases on egg production and nutrient digestibility in layers fed reduced phosphorus diets, Poult. Sci. 86 (2007) 2337–2342. [18] V. Pirgozliev, O. Oduguwa, T. Acamovic, M.R. Bedford, Effects of dietary phytase on performance and nutrient metabolism in chickens, Br. Poult. Sci. 49 (2008) 144–154. [19] S.C. Lin, W.T. Liu, S.H. Liu, W.I. Chou, B.K. Hsiung, I.P. Lin, C.C. Sheu, M. Dah-Tsyr Chang, Role of the linker region in the expression of Rhizopus oryzae glucoamylase, BMC Biochem. 8 (2007) 9. [20] T.Y. Fang, L.L. Lin, W.H. Hsu, Recovery of isoamylase from Pseudomonas amyloderamosa by adsorption–elution on raw starch, Enzyme Microb. Technol. 16 (1994) 247–252. [21] L.L. Lin, T.Y. Fang, W.S. Chu, W.H. Hsu, Improved elution of isoamylase adsorbed on raw starch and the preservation of purified enzyme, Lett. Appl. Microbiol. 19 (1994) 383–385. [22] K. Terpe, Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems, Appl. Microbiol. Biotechnol. 60 (2003) 523–533. [23] A. Jungbauer, About the cover: the Z basic protein tag, Biotechnol. J. 1 (2006) 113. [24] U. Srinivasan, J.A. Bell, A convenient method for affinity purification of maltose binding protein fusions, J. Biotechnol. 62 (1998) 163–167. [25] D. Sachdev, J.M. Chirgwin, Fusions to maltose-binding protein: control of folding and solubility in protein purification, Methods Enzymol. 326 (2000) 312–321. [26] P. Szweda, R. Pladzyk, R. Kotlowski, J. Kur, Cloning, expression, and purification of the Staphylococcus simulans lysostaphin using the intein–chitin-binding domain (CBD) system, Protein Expr. Purif. 22 (2001) 467–471. [27] H.B. Huang, M.C. Chi, W.H. Hsu, W.C. Liang, L.L. Lin, Construction and one-step purification of Bacillus kaustophilus leucine aminopeptidase fused to the starch-binding domain of Bacillus sp. strain TS-23 alpha-amylase, Bioprocess. Biosyst. Eng. 27 (2005) 389–398.
Contents lists available at ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
CBM21 starch-binding domain: A new purification tag for recombinant protein engineering Shu-Chuan Lin a, I-Ping Lin a, Wei-I Chou b, Chen-An Hsieh a, Shi-Hwei Liu b, Rong-Yuan Huang a, Chia-Chin Sheu b, Margaret Dah-Tsyr Chang a,* a b
Institute of Molecular and Cellular Biology, Department of Life Science, National Tsing Hua University, Hsinchu 300, Taiwan, ROC Simpson Biotech Co., Ltd., Taoyuan County 333, Taiwan, ROC
a r t i c l e
i n f o
Article history: Received 10 December 2008 and in revised form 12 January 2009 Available online 23 January 2009 Keywords: Rhizopus oryzae Glucoamylase Starch-binding domain Enhanced green fluorescent protein Protein purification Carbohydrate-binding module
a b s t r a c t The use of protein fusion tag technology simplifies and facilitates purification of recombinant proteins. In this article, we have found that the starch-binding domain derived from Rhizopus oryzae glucoamylase (RoSBD), a member of carbohydrate-binding module family 21 (CBM21) with raw starch-binding activity, is favorable to be applied as an affinity tag for fusion protein engineering and purification in Escherichia coli and Pichia pastoris systems. To determine suitable spatial arrangement of RoSBD as a fusion handle, enhanced green fluorescent protein (eGFP) was fused to either the N- or C-terminus of the SBD, expressed by E. coli, and purified for yield assessment and functional analysis. Binding assays showed that the ligand-binding capacity was fully retained when the RoSBD was engineered at either the N-terminal or the C-terminal end. Similar results have been obtained with the RoSBD-conjugated phytase secreted by P. pastoris. The effective adsorption onto raw starch and low cost of starch make RoSBD practically applicable in terms of development of a new affinity fusion tag for recombinant protein engineering in an economic manner. Ó 2009 Elsevier Inc. All rights reserved.
In nature, many amylolytic enzymes, such as a-amylase, b-amylase, and glucoamylase (GA),1 possess specific raw starchbinding ability through their carbohydrate-binding modules (CBMs). CBMs are defined protein domains capable of binding polysaccharide moieties. To date within 53 CBM families, eight representative members are characterized to possess starch-binding activities and thus named as starch-binding domains (SBDs). Among which the N-terminal SBD of Rhizopus oryzae glucoamylase (RoSBD) belongs to CBM family 21 and shares a relatively low level of similarity with the SBDs derived from the other starch-processing enzymes. RoSBD is a functional ligand-binding domain and has been demonstrated to be effectively adsorbed onto raw starch and other soluble oligosaccharides [1,2]. It has a molecular weight of 12 kDa and is classified as a member of Type B glycan chain-binding CBM [3]. The location and orientation of the aromatic residues in the binding sites of Type B CBMs are discovered to serve as the major factors in determining ligand affinity and specificity [4,5]. We have previously demonstrated that RoSBD possesses two ligand-binding sites, where hydrophobic interactions between the sugar rings and six aromatic
* Corresponding author. Fax: +886 3 5715934. E-mail address: [email protected] (M.D. Chang). 1 Abbreviations used: GA, glucoamylase; CBM, carbohydrate-binding module; SBD, starch-binding domain; MBP, maltose-binding protein; eGFP, enhanced green fluorescent protein; CBD, carbohydrate-binding domain; WSSV, white spot syndrome virus; HIV, human immunodeficiency virus. 1046-5928/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2009.01.008
residues on RoSBD surface play crucial roles. Specifically the residues Tyr32, Phe58, and Tyr67 constitute the binding site I, whereas the residues Trp47, Tyr83, and Tyr94 form the binding site II [2]. A large repertoire of protein purification tags is now available [6], such as polyhistidine (Poly-His), maltose-binding protein (MBP) and glutathione S-transferase (GST) [7–9]. The major obstacle that hinders these tags to be developed into large-scale bioprocessing technique is the cost of the affinity resin and other required reagents. In this paper, we describe the procedures for purifying SBD-fusion proteins based on raw starch adsorption–elution method. Raw starch is a suitable material for affinity separation of proteins due to its abundance, stability, nontoxicity, and easy recovery [10,11], hence large-scale manufacture process using raw starch seems very feasible. Our RoSBD possesses a high binding affinity towards raw starch [1]. Starch is an abundant polysaccharide composed of two types of D-glucose polymers: amylose and amylopectin. The former is composed of mostly linear a-1,4-linked glucose residues, whereas the latter is the highly branched component of starch containing 5–6% of a-1,6-linkages [12]. We use two different heterologous expression systems, Escherichia coli and Pichia pastoris to demonstrate the performance of the recovery system. For E. coli expression system, enhanced green fluorescent protein (eGFP) [13] was used as the target protein because its fluorescence could be observed only when it was correctly folded [14]. For P. pastoris expression system, phytase [15,16], an enzyme in poultry feed to lower phosphorus levels in poultry litter [17,18], was se-
262
S.-C. Lin et al. / Protein Expression and Purification 65 (2009) 261–266
lected as a candidate for secretory protein expression and purification. Materials and methods Strains, media, and plasmids Escherichia coli TOP10F’ (Invitrogen) was used for plasmid manipulation, while the E. coli strain BL21-CodonPlusÒ (DE3) (Stratagene) and the P. pastoris strain GS115 were used for protein expression. E. coli cells were grown in Luria–Bertani (LB) medium (1% tryptone, 0.5% yeast extract, and 0.5% sodium chloride) containing 50 lg/ml ampicillin at 37 °C. P. pastoris cells were grown in yeast/peptone/dextrose medium (YPD; 1% yeast extract, 2% peptone, and 2% dextrose) or buffered glycerol-complex (BMGY; 1% yeast extract, 2% peptone, 1% glycerol, 1.34% YNB, 4 10 5%, 100 mM potassium phosphate, pH 6.0) medium at 30 °C. The plasmid vectors used in this work were as follows: pGEMÒ-T Easy vector (Promega), pET23a (+) (Novagen), pPIC9 (Invitrogen), and pPICZaA (Invitrogen). Construction of the recombinant plasmids The fragments encoding RoSBD, eGFP, and phytase were individually amplified by PCR following standard protocols (Table 1). Amplified products were analyzed on 1% agarose gel, purified and ligated to pGEM-T vector, and verified by DNA sequencing. These fragments were then individually ligated into E. coli or P. pastoris expression vectors. Expression of fusion protein in E. coli The pET expression vector was transformed into BL21-CodonPlusÒ (DE3) using heat shock method. Protein expression was induced by addition of 0.5 mM IPTG to the cultures and incubation at 20 °C for 16 h. Cells were harvested by centrifugation, washed and then disrupted by sonication. Sonicated suspensions were centrifuged at 10,000g for 30 min at 4 °C and the supernatants were sterile filtered (0.2 lm). Expression of fusion protein in P. pastoris Transformation of Pichia was performed by lithium chloride method according to the manufacturer’s protocol (Invitrogen). Ten micrograms of the recombinant plasmid, pPICZaA/sbd-phytase or pPIC9/phytase-sbd was linearized using restriction enzyme PmeI and electroporated into P. pastoris GS115. Positive colonies
were selected in YPD plates containing 100 lg/mL Zeocine and sequentially induced for recombinant protein expression by culturing them in BMGY, followed by buffered methanol-complex medium (BMMY) containing 0.75% methanol. After 72 h, culture supernatants were collected by centrifugation at 3000g for 30 min at 4 °C. Purification of SBD and SBD-fusion proteins RoSBD was purified using prepacked HiTrapTM SP column [19]. All SBD-fusion proteins were purified by affinity chromatography using an amylose resin column. A 2-mL bed of amylose resin (New England Biolabs) was poured into a 1.5 10 cm column, equilibrated in buffer (50 mM NaOAc, pH 5.5), and the clarified cell lysate containing the SBD-fusion protein was loaded onto the column. The column was then washed with binding buffer (50 mM NaOAc, pH 5.5) and the adsorbed proteins were eluted with elution buffer (10 mM glycine/NaOH, pH 11). In addition, two purification methods were developed using raw starch in this study. One was stirring method, referred to Fang’s method [20]. Fifty milligrams corn starch (Sigma–Aldrich, EC 232-679-6) were washed three times in elution buffer and binding buffer as described above. Prewashed starch was suspended in 1 mL protein solution with slow rotation on a platform shaker at 25 °C for 3 h. The starch pellet with adsorbed proteins was washed with 3 mL of binding buffer, and then eluted by 1.5 mL elution buffer. The other was columnbased method developed by modifying a method proposed by Lin [21]. The corn starch (200 mg) was washed as described for the stirring method and packed into a 5-mL column in which a filter paper was placed at the bottom. After the protein solution (3 mL) was loaded onto the column, the column was washed with 6 mL binding buffer and the adsorbed proteins were eluted with 3 mL elution buffer. The protein purity was determined by 12% or 15% SDS–PAGE and protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent kit (Pierce), with BSA as the reference standard. Association rate assay of SBD The purified SBD in the concentration range from 15.6 to 27.2 lM was mixed with prewashed corn starch (1 mg/mL) in a final volume of 100 lL and incubated at 25 °C with gentle stir for 5 h. The binding was terminated at different time intervals by sedimentation of the starch. After centrifugation at 16,000g for 10 min at 4 °C, the protein concentration in the supernatant (unbound protein) was determined by the BCA assay, and the amount of bound protein was calculated from the difference between the ini-
Table 1 Plasmids generated and used in this study. Name of the plasmids
Description
Ref.
pET23a/sbd pET23a/egfp–sbd
The sbd gene was cloned into the NdeI/XhoI-restricted pET23a vector. The cloned egfp and sbd were excised with EcoRI/HindIII and HindIII/XhoI and inserted together into the EcoRI and XhoI sites of pET23a vector. The cloned sbd and egfp were excised with NdeI/KpnI and KpnI/XhoI and inserted together into the NdeI and XhoI sites of pET23a vector. The cloned sbd-l58 and egfp were excised with NdeI/KpnI and KpnI/XhoI and inserted together into the NdeI and XhoI sites of pET23a vector. The cloned sbd-l38 and egfp were excised with NdeI/KpnI and KpnI/XhoI and inserted together into the NdeI and XhoI sites of pET23a vector. The cloned sbd-l8 and egfp were excised with NdeI/KpnI and KpnI/XhoI and inserted together into the NdeI and XhoI sites of pET23a vector. The gene fragment sbd-phytase was generated by two-step PCR and the amplified fragment was digested using EcoRI and inserted into the Pichia expression vector pPICZaA. The gene fragment phytase-sbd was generated by two-step PCR and the amplified fragment was digested using KpnI/EcoRI and inserted into the Pichia expression vector pPIC9.
[2,3,14] This study
pET23a/sbd–egfp pET23a/sbd-l58– egfp pET23a/sbd-l38– egfp pET23a/sbd-l8– egfp pPICZaA/sbdphytase pPIC9/phytase-sbd
This study This study This study This study This study This study
S.-C. Lin et al. / Protein Expression and Purification 65 (2009) 261–266
tial and unbound protein concentrations. The bound protein at equilibrium expressed as micromole of protein per gram of starch was a linear function of the free (unbound) protein in the range of protein concentrations assayed. Saturation binding assay The purified SBD or SBD-fusion protein at a concentration ranging from 10 to 120 lM was mixed with 1 mg of prewashed corn starch in 50 mM NaOAc, pH 5.5 and incubated at 25 °C for 3 h. After centrifugation at 16,000g for 10 min at 4 °C, the unbound protein was determined by BCA assay. The amount of adsorbed protein was calculated from the difference between the initial and unbound protein concentrations. Kinetic parameters were determined from the binding isotherms with non-linear regression (GraphPad Prism 4). Effects of pH on binding ability The purified protein with a concentration of 20 lM (0.25 mg/ mL) was added to 100 lL of prewashed corn starch (1 mg/mL) in buffer solution with different pH values and incubated at 25 °C for 1 h. The buffers used were 50 mM glycine/HCl (pH 2–3), sodium acetate/acetic acid (pH 4–5.5), Na2HPO4/NaH2PO4 (pH 6–7), Tris/ HCl (pH 8), and glycine/NaOH (pH 9–11). The free fusion protein concentration of the supernatant before and after the binding was determined by BCA assay. The relative binding ability of the fusion protein assayed at pH 5.5 was normalized as 100%.
263
Effects of starch on SBD adsorption One hundred milligrams of raw starch from various sources were prewashed and suspended in 1 mL binding buffer (50 mM NaOAc, pH 5.5). Thirty microliters (3 mg) of starch solution were added to 90 lL of protein solution (final concentration of 1 mg/mL), and the suspension was incubated at 25 °C with gentle stirring for 2 h. After incubation, the samples were centrifuged at 16,000g for 10 min at 4 °C, the unbound protein concentration of the supernatant was determined and the adsorption percentage was calculated. Results and discussion Characteristic properties of RoSBD The RoSBD structure revealed eight b-strands arranged essentially in an antiparallel fashion, forming an open-sided b-barrel organized into two b-sheets (Fig. 1A) [2,12]. As illustrated in Fig. 1A, the key aromatic residues in the ligand-binding sites I and II were indicated. The pH effects on the binding of RoSBD to corn starch indicated that the protein was able to tightly bind to raw starch at a pH range from 4.0 to 7.0. Such ligand binding of RoSBD appeared to be pH dependent, as the maximal binding was gained at pH 4.0 and pH 5.0, whereas the binding was significantly weakened at the pH values below 4.0 and above 8.0 (Fig. 1B). In order to determine the time needed to reach equilibrium for binding to starch, a time-course experiment was performed. In the initial period (up to 50 min), the adsorption exhibited a linear phase and the
Fig. 1. Characterization of RoSBD. (A) The structure of apo RoSBD (PDB code 2VQ4) with eight b-strands forming two b-sheets and a distorted barrel. (B) pH effect on the adsorption of RoSBD to corn starch. The binding ability of RoSBD at varying pH was measured as described in Materials and methods. (C) Time course of adsorption of RoSBD to corn starch. Purified protein at different concentrations (N, 15.6; j, 22.5, and d, 27.2 lM) was added to 0.1 mg of prewashed granular corn starch and incubated at 25 °C with gentle stirring for 5 h. (D) Adsorption isotherm of RoSBD binding to corn starch at pH 5.5 and 25 °C.
264
S.-C. Lin et al. / Protein Expression and Purification 65 (2009) 261–266
maximal binding was achieved after 2 h (Fig. 1C). Furthermore, the adsorption isotherm of RoSBD resulted in a Bmax of 45.5 ± 1.4 lmol/ g and Kd of 6.9 ± 0.7 lM (Fig. 1D). Purification of RoSBD by amylose affinity chromatography The adsorption of RoSBD to raw starch was strongly dependent on the pH, suggesting that electrostatic interactions play an important role in the adsorption process. Hence the recombinant RoSBD was affinity purified by one-step chromatography on cross-linked amylose (Fig. 2A), where the binding and elution steps were performed in buffers with pH values of 5.5 (50 mM NaOAc) and 11.0 (10 mM glycine/NaOH), respectively. The adsorption capacity of amylose resin was greater than 50 mg/mL, and the adsorbed SBD was eluted with the basic buffer to give high purity SBD exceeding 97% with this single-step purification scheme. An average yield of 30 mg purified RoSBD could be obtained from 1 L E. coli culture. RoSBD as a purification handle in E. coli expression system In order to evaluate the application of the RoSBD as a purification handle, recombinant proteins consisting SBD fused to eGFP were expressed in E. coli system. Both N- and C-terminal fusions could be produced and readily purified by the procedure as described above. Similar to the case of RoSBD, the 39 kDa recombinant SBD-fusion proteins with high purities exceeding 90% could be obtained (Fig. 2B). The system has a high capacity to purify recombinant fusion proteins in E. coli because the background of endogenous protein interacting with starch is quite low. These results confirmed that RoSBD could confer starch-binding activity in both N- and C-terminal fusions.
Fig. 3. Effects of various starches on the adsorption of SBD and SBD-fusion proteins. Three milligrams of starch was used for adsorbing the target protein at a concentration of 1 mg/mL in a total volume of 120 lL. After incubation at 25 °C for 2 h, the sample was centrifuged and the adsorption percentage was determined. Each data point represents the average of three independent measurements, and error bars represent SD.
Purified SBD–eGFP and eGFP–SBD were subsequently used to evaluate the application of native starch as an affinity adsorbent. To investigate the effects of starch from a variety of natural sources on the purification, each native starch was prepared by washing with 10 mM glycine/NaOH (pH 11) and 50 mM NaOAc (pH 5.5). The fusion protein was separately added to the washed starch (3 mg) in a total volume of 120 lL with initial protein concentration of 1 mg/mL, and the mixture was stirred at 25 °C for 2 h. In comparison with the adsorption percentage of SBD, SBD–eGFP, and eGFP–SBD with different kinds of starch, Fig. 3 revealed that the adsorption efficiency of corn, rice, sweet potato, tapioca, and wheat starch was greater than 90%, while that of the other three starches from mung bean, potato and soybean was less than 60%. Although corn, rice, sweet potato, tapioca, and wheat starch had comparable adsorption capacity, corn starch was chosen as the adsorbent for the following studies due to its lower cost. In addition, the SBD and the targeted eGFP fusion proteins showed similar properties in terms of starch binding. Purification of SBD-tagged fusion proteins by corn starch
Fig. 2. Purification of SBD using amylose resin. (A) E. coli cells from 200 mL of culture were collected by centrifugation. After lysis, cells were centrifuged to remove cell debris, and then loaded onto the amylose resin pre-equilibrated with binding buffer 50 mM NaOAc, pH 5.5. After wash with 30 mL equilibration buffer, 10 mM NaOAc, pH 11.0 was added to elute SBD and 10 mL fractions were collected. Lanes represent the column load (L), flow through (F), and wash (W), followed by four lanes of eluate (E). Samples (20 lL) were separated by 15% SDS–PAGE and stained with Coomassie blue. (B) SDS–PAGE (15%) analysis of purified SBD and SBDfusion proteins. SBD, SBD–eGFP, and eGFP–SBD were expressed in E. coli and the fusion proteins SBD-Phytase and Phytase-SBD were expressed and purified from the P. pastoris culture medium. Lanes 1, SBD; 2, SBD–eGFP; 3, eGFP–SBD; 4, SBDPhytase; and 5, Phytase-SBD.
Together with the application of natural starch, corn starch with the required properties was selected as a model to study its effectiveness. Here stirring and column-based methods were developed for protein purification. Three different fusion proteins with interdomain linkers of 8, 38, and 58 amino acid residues, named SBDL8–eGFP, SBD-L38–eGFP, and SBD-L58–eGFP, respectively, were tested. The characteristic starch-binding properties of SBD-L8– eGFP were quite similar to that of RoSBD (Supplementary Fig. 1). The affinity of SBD-L8–eGFP to corn starch was 2.7 ± 0.2 lM and the Bmax value was determined as 77.6 ± 1.5 lmol/g. Table 2 summarized the data from those SBD-fusion proteins with regard to the purification efficiency in terms of adsorption and recovery rate. The adsorption (%) is defined as [(original protein concentration supernatant protein concentration)/original protein concentration] 100. The recovery rate (%) is defined as (eluted protein
S.-C. Lin et al. / Protein Expression and Purification 65 (2009) 261–266 Table 2 Purification methods for recombinant SBD-fusion proteins. Protein samples
Adsorption (%)a
Recovery rate (%)b
Stirring-based method SBD-L58–eGFP SBD-L38–eGFP SBD-L8–eGFP
90.3 ± 1.2 94.6 ± 2.4 96.6 ± 6.7
50.0 ± 5.7 55.4 ± 11.9 44.9 ± 9.9
Column-based method SBD-L58–eGFP SBD-L38–eGFP SBD-L8–eGFP
84.7 ± 8.3 84.5 ± 12.0 82.0 ± 5.4
83.6 ± 9.2 86.0 ± 2.5 87.1 ± 4.1
The data are expressed as average value ± SD of three independent trials. a The adsorption (%) is defined as [(original protein concentration supernatant protein concentration)/original protein concentration] 100. b The recovery rate (%) is defined as (eluted protein concentration/adsorbed protein concentration) 100.
concentration/adsorbed protein concentration) 100. As an illustration, stirring method (Fig. 4A) for the preparation of adsorbent had high adsorption efficiency (90–97%), but the recovery rate was relatively low (50–55%). Alternatively, the column-based method (Fig. 4B) gave relatively lower adsorption efficiency (82– 85%), but the average recovery rate was 86%, much better than that of the stirring-based method. These results suggest that it is feasible to use corn starch as an adsorbent for the recovery and purification of recombinant fusion proteins. In addition, among the three SBD-fusion proteins with linker variants used in this study, insertion of the linker region between RoSBD and eGFP did not affect the starch-binding affinity. In comparison, although the starch adsorption rate was promoted in the stirring method, the recovery yield was significantly improved in the column-based one. Moreover, as the sample was forced to pass through a simple column packed with starch, rapid recovery of the target protein was achieved. However, the conse-
Fig. 4. Purification of SBD-fusion proteins using raw starch. The purification was carried out by (A) stirring and (B) column-based methods. Three SBD–eGFP fusion proteins with L8, L38, and L58 linkers containing 8, 38, and 58 amino acid residues were, respectively, indicated, lanes represent the size marker (M), column load (L), flow through (F), wash (W), and eluate (E). An equal volume (12 lL) of each sample was loaded onto 12% SDS–PAGE.
265
quence for lower adsorption efficiency in the system was possibly correlated with short contact time. The result indicated that the column-based method was more effective, and it could be further developed into a commercial purification technique due to the convenience, efficiency and economy. RoSBD as a purification tag in P. pastoris expression system Since E. coli is only one of several popular recombinant expression hosts, the practical application of SBD tag in P. pastoris expression system for production of secretory proteins was evaluated as well. Here E. coli phytase capable of degrading phytic acid was selected as the target protein. The column-based method was used to assess the ability of RoSBD for purification of secreted recombinant phytase (SBD-Phytase and Phytase-SBD) from the bulk yeast culture media. The absorption efficiency of these recombinant proteins (61–64%) was found to be lower than that of E. coli system (90–97%), presumably due to short contact time passing through the column or competition of oligosaccharides in the culture medium. In addition, 30–40% of the SBD-tagged proteins present in the culture media could be retrieved in the elution fraction. The purity of both SBD-Phytase and Phytase-SBD was higher than 90% according to SDS–PAGE analysis (Fig. 2B). Conclusion Affinity tags are important for facilitating protein purification [22]. Rapid and low-cost production of recombinant proteins is necessary for generation of industrially relevant proteins. Fusion protein technology has been enthusiastically pursued since the advent of genetic engineering. To add a molecular handle to a recombinant protein is an attractive way to simplify the purification scheme [23]. Many polysaccharide hydrolyzing enzymes display a carbohydrate-binding domain (CBD), and a few of them have been extensively studied for recombinant protein purification. For example, E. coli MBP is a commonly used tag for streamlined purification [23–25], but the relatively large size of MBP may preclude the structure or function of target protein. Moreover, the chitin-binding domain from Bacillus circulans was applied as the affinity tag to purify Staphylococcus simulans lysostaphin [26], but it required a high salt concentration (higher than 500 mM) or the use of non-ionic detergents to optimize the purity of the product, so this harsh condition might reduce its application potentiality. In addition, many CBDs with the size ranging from 4 to 20 kDa are currently available for purification purpose [22]. A major drawback is the stringent elution condition, which may compromise purification and further use of the tagged proteins. CBMs are now grouped into 53 families according to ligand specificity and 8 CBM family members are characterized to possess starch-binding activities. Based on the characteristics of starchbinding CBMs, starch and its derivatives have been used for the elution of a-amylase, b-amylase, glucoamylase, isoamylase, and cyclodextrin glucantransferase from the adsorbents [27]. Here the RoSBD, the first CBM21 member being structurally and functionally characterized, used in this study has relatively small size of 12 kDa and binds to starch over the pH range from 4.0 to 7.0. It can be used as either N- or C-terminal fusion partners for efficient purification. Moreover, we have also applied this system to purify two viral proteins at laboratory scale. For example, the envelope viral protein VP28 of white spot syndrome virus (VP28/WSSV) and the envelope glycoprotein protein GP41 of human immunodeficiency virus (GP41/HIV) were separately engineered with N-terminal SBD-fusion head. As expected, both proteins were successfully expressed and purified (Supplementary Fig. 2). There-
266
S.-C. Lin et al. / Protein Expression and Purification 65 (2009) 261–266
fore, a number of tested results verified that the purification method developed in this study is simple and efficient. In some applications, it may not be necessary to remove the RoSBD tag from the fusion proteins. For instance, phytase is a feed supplement to the diet of domesticated animals; using starch adsorption to recover the fusion proteins takes advantages of direct product collection, which needs no further separation. As a result, the properties of starch make it a better value for particular application. In summary, we have provided direct evidence showing that RoSBD can be attached to different proteins to facilitate purification on starch. The fusion proteins produced by E. coli and P. pastoris cells could be recovered with high purity by adsorption–elution procedure on raw starch. The RoSBD is thus demonstrated to offer a convenient, economical and easy solution to manifest recombinant protein engineering and purification. Acknowledgments This work is supported by Simpson Biotech Co., Ltd., Taiwan, ROC; National Science Council, ROC, Grant 97-2622-B-007-001; and Council of Agriculture and the National Science and Technology Program for Agricultural Biotechnology, Grant 97AS-1.2.1.ST-a5 to M. D.-T. Chang. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.pep.2009.01.008. References [1] W.I. Chou, T.W. Pai, S.H. Liu, B.K. Hsiung, M.D. Chang, The family 21 carbohydrate-binding module of glucoamylase from Rhizopus oryzae consists of two sites playing distinct roles in ligand binding, Biochem. J. 396 (2006) 469–477. [2] J.Y. Tung, M.D. Chang, W.I. Chou, Y.Y. Liu, Y.H. Yeh, F.Y. Chang, S.C. Lin, Z.L. Qiu, Y.J. Sun, Crystal structures of the starch-binding domain from Rhizopus oryzae glucoamylase reveal a polysaccharide-binding path, Biochem. J. 416 (2008) 27–36. [3] A.B. Boraston, D.N. Bolam, H.J. Gilbert, G.J. Davies, Carbohydrate-binding modules: fine-tuning polysaccharide recognition, Biochem. J. 382 (2004) 769– 781. [4] L. Guan, Y. Hu, H.R. Kaback, Aromatic stacking in the sugar binding site of the lactose permease, Biochemistry 42 (2003) 1377–1382. [5] T. Ponyi, L. Szabo, T. Nagy, L. Orosz, P.J. Simpson, M.P. Williamson, H.J. Gilbert, Trp22, Trp24, and Tyr8 play a pivotal role in the binding of the family 10 cellulose-binding module from Pseudomonas xylanase A to insoluble ligands, Biochemistry 39 (2000) 985–991. [6] M. Hedhammar, S. Hober, Z(basic)—a novel purification tag for efficient protein recovery, J. Chromatogr. A 1161 (2007) 22–28.
[7] D.B. Smith, K.S. Johnson, Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase, Gene 67 (1988) 31–40. [8] K. Terpe, Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems, Appl. Microbiol. Biotechnol. 60 (2003) 523–533. [9] M.B. Murphy, S.A. Doyle, High-throughput purification of hexahistidine-tagged proteins expressed in E. coli, Methods Mol. Biol. 310 (2005) 123–130. [10] L.J. Chen, C. Ford, A. Kusnadi, Z.L. Nikolov, Improved adsorption to starch of a beta-galactosidase fusion protein containing the starch-binding domain from Aspergillus glucoamylase, Biotechnol. Prog. 7 (1991) 225–229. [11] L.J. Chen, C. Ford, Z. Nikolov, Adsorption to starch of a beta-galactosidase fusion protein containing the starch-binding region of Aspergillus glucoamylase, Gene 99 (1991) 121–126. [12] Y.N. Liu, Y.T. Lai, W.I. Chou, M.D. Chang, P.C. Lyu, Solution structure of family 21 carbohydrate-binding module from Rhizopus oryzae glucoamylase, Biochem. J. 403 (2007) 21–30. [13] B.P. Cormack, R.H. Valdivia, S. Falkow, FACS-optimized mutants of the green fluorescent protein (GFP), Gene 173 (1996) 33–38. [14] N.J. Caron, Y. Torrente, G. Camirand, M. Bujold, P. Chapdelaine, K. Leriche, N. Bresolin, J.P. Tremblay, Intracellular delivery of a Tat–eGFP fusion protein into muscle cells, Mol. Ther. 3 (2001) 310–318. [15] C.C. Chen, P.H. Wu, C.T. Huang, K.J. Cheng, A Pichia pastoris fermentation strategy for enhancing the heterologous expression of an Escherichia coli phytase, Enzyme Microb. Technol. 35 (2004) 315–320. [16] H. Luo, H. Huang, P. Yang, Y. Wang, T. Yuan, N. Wu, B. Yao, Y. Fan, A novel phytase appA from Citrobacter amalonaticus CGMCC 1696: gene cloning and overexpression in Pichia pastoris, Curr. Microbiol. 55 (2007) 185–192. [17] N. Liu, G.H. Liu, F.D. Li, J.S. Sands, S. Zhang, A.J. Zheng, Y.J. Ru, Efficacy of phytases on egg production and nutrient digestibility in layers fed reduced phosphorus diets, Poult. Sci. 86 (2007) 2337–2342. [18] V. Pirgozliev, O. Oduguwa, T. Acamovic, M.R. Bedford, Effects of dietary phytase on performance and nutrient metabolism in chickens, Br. Poult. Sci. 49 (2008) 144–154. [19] S.C. Lin, W.T. Liu, S.H. Liu, W.I. Chou, B.K. Hsiung, I.P. Lin, C.C. Sheu, M. Dah-Tsyr Chang, Role of the linker region in the expression of Rhizopus oryzae glucoamylase, BMC Biochem. 8 (2007) 9. [20] T.Y. Fang, L.L. Lin, W.H. Hsu, Recovery of isoamylase from Pseudomonas amyloderamosa by adsorption–elution on raw starch, Enzyme Microb. Technol. 16 (1994) 247–252. [21] L.L. Lin, T.Y. Fang, W.S. Chu, W.H. Hsu, Improved elution of isoamylase adsorbed on raw starch and the preservation of purified enzyme, Lett. Appl. Microbiol. 19 (1994) 383–385. [22] K. Terpe, Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems, Appl. Microbiol. Biotechnol. 60 (2003) 523–533. [23] A. Jungbauer, About the cover: the Z basic protein tag, Biotechnol. J. 1 (2006) 113. [24] U. Srinivasan, J.A. Bell, A convenient method for affinity purification of maltose binding protein fusions, J. Biotechnol. 62 (1998) 163–167. [25] D. Sachdev, J.M. Chirgwin, Fusions to maltose-binding protein: control of folding and solubility in protein purification, Methods Enzymol. 326 (2000) 312–321. [26] P. Szweda, R. Pladzyk, R. Kotlowski, J. Kur, Cloning, expression, and purification of the Staphylococcus simulans lysostaphin using the intein–chitin-binding domain (CBD) system, Protein Expr. Purif. 22 (2001) 467–471. [27] H.B. Huang, M.C. Chi, W.H. Hsu, W.C. Liang, L.L. Lin, Construction and one-step purification of Bacillus kaustophilus leucine aminopeptidase fused to the starch-binding domain of Bacillus sp. strain TS-23 alpha-amylase, Bioprocess. Biosyst. Eng. 27 (2005) 389–398.