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A linker peptide with high affinity towards silica-containing materials
New Biotechnology Volume 30, Number 5 June 2013
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Research Paper
A linker peptide with high affinity towards silica-containing materials Anwar Sunna1, Fei Chi1 and Peter L. Bergquist1,2 1 2
Department of Chemistry and Biomolecular Sciences, and Environmental Biotechnology CRC, Macquarie University, Sydney, Australia Department of Molecular Medicine & Pathology, University of Auckland Medical School, Auckland, New Zealand
A peptide sequence with affinity to silica-containing materials was fused to a truncated form of Streptococcus strain G148 Protein G. The resulting recombinant Linker-Protein G (LPG) was produced in Escherichia coli and purified to apparent homogeneity. It displayed high affinity towards two natural clinoptilolite zeolites. The LPG also displayed high binding affinity towards commercial-grade synthetic zeolite, silica and silica-containing materials. A commercial sample of the truncated Protein G and a basic protein, both without the linker, did not bind to natural or synthetic zeolites or silica. We conclude that the zeolite-binding affinity is mediated by the linker peptide sequence. As a consequence, these data may imply that the binding affinity is directed to the SiO2 component rather than to the atomic orientation on the zeolite crystal surface as previously assumed.
Introduction The interaction of proteins with solid surfaces is central to several chromatographic purification and biosensor techniques [1]. The application of unmodified silica in protein purification has been used relatively rarely [2]. More common are methods in which a biological recognition molecule is immobilized onto a silica or silica-based sensing substrate. Methods involving covalent immobilization are generally achieved in three steps: modification of the surface to add specific functional groups, covalent attachment of a crosslinker through one of its reactive moieties and covalent linking of the biomolecular recognition element to the remaining reactive sites of the crosslinker. Methods have been described to allow immobilization of biological recognition molecules onto sensing surfaces with full functionality in biosensor-binding assays. A frequent theme has been the orientated immobilization of antibodies on silicon wafers [3,4] or nanowires as electronic devices for sensitive and direct detection of biological analytes [5]. Significant progress has been made on a range of immobilization supports [6,7]. However, the immobilization of proteins has been a particularly challenging task, mainly due to their heterogeneous nature and the marginal stability of the active tertiary Corresponding author: Sunna, A. ([email protected])
structure over the denatured (and inactive) random coil structure. Most of the methods available for immobilizing proteins onto solid supports traditionally have relied on nonspecific adsorption [8,9] or on the reaction of naturally occurring chemical groups such as amines and carboxylic acids within proteins with appropriate reactive groups on the matrix [10,11]. In both cases, the corresponding proteins are attached to the surface in a random orientation that may cause the reduction or loss of the protein’s biological activity [12]. Recent reviews have summarized progress and the status of the field [7,13]. The techniques described above are elegant but may not lend themselves to inexpensive bulk applications particularly suited to field conditions. Zeolites are inorganic materials that have a highly ordered structure and can be synthesized as nanocrystals with pores and channel systems in the molecular size range of 0.3–3.0 nm. They offer interesting characteristics as inorganic support materials to immobilize proteins including mechanical and chemical resistance and high surface area [14,15]. The basic/acidic nature of zeolites can be modified by varying the Si/Al ratio or by introducing different metals into the crystalline framework and by changing the Si/metal ratio [16]. The acidity of zeolites can be modified by exchanging extra-framework metal cations with H+ ions. They are known to be stable both in wet and dry conditions.
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Brown [17–19] reported that repeating polypeptides fused to protein sequences could bind to their target metals with high affinity and specificity. Nygaard et al. [20] selected specific zeolitebinding proteins by Escherichia coli cell-surface display. The candidate sequences for zeolite-binding proteins were transferred from the gene encoding the bacterial surface protein (lamB) and fused to the alkaline phosphatase (phoA) gene. The soluble fusion protein expressed was shown to bind to zeolite and displayed enzymatic activity. One sequence described for plasmid pSN6 coded for 21 amino acid residues (VKTQATSREEPPRLPSKHRPG) repeated four times plus another 7 residues of a partial 5th repeat [20]. The fusion protein was reported to bind specifically only to two synthetic zeolites, Faujasite (FAU) and EMT (both synthesized by SINTEF, Norway) but not to other zeolites. We reasoned that other materials containing silica might be bound by the same or a related linker sequence that permits specific binding to zeolite. We fused the above linker to a truncated form of Streptococcus Protein G, Protein G’, [21] and examined the ability of the resulting recombinant protein to bind to silica-containing materials. In this work we present a detailed and extensive examination of the binding affinity of this recombinant protein.
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Production and purification of recombinant LPG
CBV and CP zeolite series were purchased from Zeolyst International (Conshohocken, PA, USA), Valfor 100 zeolite was provided by PQ Australia Pty Ltd. (Dandenong South, VIC, Australia), molecular sieves 5A and 13X were from Fujian Anten Chemical Co. Ltd. (Xiamen, China), clinoptilolite samples were provided by Zeolite Australia Pty Ltd. (Werris Creek, NSW, Australia) and Castle Mountain Zeolites Pty Ltd. (Quirindi, NSW, Australia). Precipitated silica was from BDH Ltd., Davisil silica grade LC 60A and 646, silica Perkasil, Elfadent and Durafill were supplied by Grace Davison Discovery Sciences (Rowville, VIC, Australia), Scharlau silica gel 60 was obtained from Chem-Supply Pty Ltd. (Adelaide, SA, Australia), silica Rhodoline and Tixosil from Rhodia were provided by Brenntag Pty Ltd. (Notting Hill, VIC, Australia).
For the production of recombinant LPG, 300 ml Luria Bertani (LB) medium supplemented with 50 mg/ml carbenicillin was inoculated with 3 ml of an overnight culture of E. coli Tuner (DE3) cells (Novagen) harbouring pOPT_LPGpET22b. The culture was incubated at 37 8C with shaking (250 rpm) until the A600 was approximately 0.7–1.0. The incubation temperature was reduced to 20 8C and protein synthesis was induced by the addition of 0.2 mM isopropyl b-D-thiogalactoside (IPTG). Cells were harvested after 3– 4 h induction by centrifugation for 15 min at 10,000 g and 4 8C and were stored at 20 8C. The cells were resuspended in ice-cold lysis buffer (25 mM Tris– HCl, pH 8.0, 100 mM NaCl, 1.25 mM EDTA and 0.05% Tween 20), supplemented with 4 mM of the Pefabloc serine protease inhibitor (Roche) and 1.5 mg lysozyme (Sigma). They were ruptured by three passages through a French pressure cell. After cell rupture, 50 U of DNase I (Invitrogen) and 4 mM Pefabloc were added. The sample was incubated with rotation at room temperature for 20 min. The debris was removed by centrifugation for 30 min at 20,000 g and 4 8C. The supernatant obtained was first filtered through a 0.45 mm and then through a 0.2 mm sterile filter and stored at 4 8C. The extract was loaded onto a 5 ml HiTrap Q anion exchanger column (GE Healthcare) previously equilibrated with 25 mM Tris– HCl, pH 8.0, supplemented with 100 mM NaCl. The column was washed extensively with the same buffer. Under these conditions the LPG was found in the 100 mM NaCl fraction. The LPG fraction was applied to a 5 ml HiTrap SP cation exchanger column (GE Healthcare) previously equilibrated with buffer containing 100 mM NaCl. The column was washed extensively with the same buffer and the LPG was eluted at 150 and 200 mM NaCl. Fractions containing the LPG were identified on a NuPAGE 4–12% Bis–Tris Gel (1.5 mm, 10 well, Invitrogen) by SDS-PAGE and staining with Coomassie Brilliant Blue and pooled and concentrated using an Amicon Ultra-15 centrifugal filter (10 kDa cut-off, Millipore). Samples were stored in 25 mM Tris–HCl, pH 8.0, 100 mM NaCl at 20 8C after sterile filtration through a 0.1 mm filter.
Construction of a Linker-Protein G (LPG) recombinant protein
Minimal number of repeats required for binding
The Protein G sequence used was based on that reported by Goward et al. [21] which codes for a truncated recombinant form of Streptococcus strain G148 Protein G (Protein G’). A DNA sequence encoding the Linker amino acid sequence (VKTQATSREEPPRLPSKHRPG)4VKTQTAS and a DNA sequence encoding a truncated Protein G’ DNA sequence were joined with the linker sequence positioned at the N-terminus of Protein G’. The codon usage of the final combined Linker and Protein G’ sequence was optimized for expression in E. coli. Rare codons and negative cisacting motifs (such as internal RBS sites, TATA-boxes, Chi-recombination sites, and so on) that can hamper expression in E. coli were removed during codon optimization. Codon optimization and gene synthesis of the final combined sequence was performed by GENEART AG (Regensburg, Germany). The final optimized sequence (OPT_LPG) was designed with flanking restriction sites (NdeI and BamHI) for ligation into plasmid pET22b (Novagen) to give the expression plasmid pOPT_LPGpET22b. The protein expressed from the codon-optimized OPT_LPG sequence is referred to here as Linker-Protein G (LPG).
Several truncated derivatives of pOPT_LPGpET22b (Fig. 1a) were prepared to determine the minimum number of repeat sequences required for binding of LPG to silica-containing materials. DNA fragments encoding the different truncated recombinant proteins were amplified by PCR using the primer pairs described in Fig. 1b. The amplified DNA fragments were digested with NdeI/BamHI and ligated into similarly cut pET22b. Production and purification of the different truncated recombinant LPG proteins was as described above.
Materials and methods Zeolite and silica
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Standard binding assay The binding of purified recombinant LPG to silica-containing material (e.g., natural and synthetic zeolite and silica) was determined as follows. Silica-containing materials were washed three times with washing buffer (10 mM Tris–HCl, pH 7.5, 100 mM NaCl and 1% Triton-X100). Soluble protein in a final volume of 100 ml was mixed with silica-containing material and incubated by rotation at room temperature for 1 h. The unbound fraction was removed after centrifugation at 14,000 g for 20 s. The pellet of
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New Biotechnology Volume 30, Number 5 June 2013
FIGURE 1
Truncated derivatives used in this study. (a) Plasmids, primers and architecture for each truncated derivative. (b) Oligonucleotides used in this study. aNumber of linker sequence repeats; bN.A, not applicable; cpurified recombinant truncated Protein G’ (Sigma–Aldrich); dengineered restriction sites are underlined.
silica-containing material was washed three times by vortexing with 100 ml of 100 mM Tris–HCl buffer, pH 8.0. The bound protein was eluted after addition of 100 ml of SDS PAGE-loading buffer and incubation at 99 8C for 10 min (with short mixing every 2 min). Fractions containing the protein were identified by SDS-PAGE and staining with Coomassie Brilliant Blue. In addition, a control-binding assay was performed with LPG and two other proteins lacking the linker sequence. Truncated Protein G’ (without the linker, Sigma–Aldrich) and a basic Dictyoglomus XynB xylanase [22] were used as controls for the binding experiments. Binding assays with Dictyoglomus XynB at different pH values were performed to assess the effect of the overall protein charge on adsorption to silica-containing materials. Dictyoglomus XynB, binding and washing buffers were adjusted to the final pH of the experiment. The following buffers at a final concentration of 100 mM each were used in the assay: Na-acetate buffer (pH 4 and 5); 2-(N-Morpholino) ethanesulfonic acid buffer (MES, pH 6); Tris buffer (pH 7–9). The percentage of protein in the different fractions was semiquantified by analysing the intensity of bands of digital images from SDS-PAGE with ImageJ software (http://rsb.info.nih.gov/ij/ index.html).
Results Purification of recombinant LPG The expression plasmid pOPT_LPGpET22b was introduced into E. coli Tuner (DE3) cells and recombinant protein production and purification were carried out as described in the ‘Materials and methods’ section. The optimized recombinant LPG protein from expression in E. coli was purified to electrophoretic homogeneity as evaluated by SDS-PAGE (data not shown). The final yield of pure LPG protein from 300 ml of medium was approximately 20 mg.
Affinity of LPG towards silica-containing materials Natural zeolite Two types of natural zeolites were tested (Fig. 2). Zeolite Australia is a zeolite comprising a 54% clinoptilolite content while Castle
FIGURE 2
Binding affinity of purified LPG to Castle Mountain and Zeolite Australia natural clinoptilolite (5 mg each). Binding was performed as described in ‘Materials and methods’ section. Wash fractions (3 100 ml) were not loaded onto the gels. S, starting LPG (80 mg); U, unbound LPG fraction; B, zeolitebound LPG fraction; LPG, Linker-Protein G.
Mountain zeolite contains 85% clinoptilolite. The cationexchange capacity (CEC) for Zeolite Australia and Castle Mountain zeolites are reported as 1.19 and 1.47 meq/g, respectively (http:// www.zeolitemineral.com/worldwide.php, March 2012). The Zeolite Australia zeolite (7 Mohs) is much harder than the Castle Mountain zeolite (5 Mohs). The purified LPG showed affinity to both types of natural zeolites (particle size <10 mm to 2.0 mm). Most of the LPG was found in the bound fraction when incubated with zeolites with particle size <10 mm to 0.5 mm. However, this affinity decreased with zeolite particles above 0.5 mm. This might be a result of larger particles having less surface area available for LPG binding. The differences in the degree of binding due to the particle size could be overcome by increasing the amount of zeolite used in the experiments (data not shown). This finding probably supports the observation that the differences are a consequence of the reduced surface area availability in larger zeolite particles.
Synthetic zeolite Thirteen synthetic zeolites belonging to 8 different zeolite families were tested as binding substrates for the purified LPG. Supplementary Table 1 summarizes some properties of the synthetic zeolites tested. This comprehensive characterization of the linker peptide (LPG) showed that the two zeolites originally reported in the Nygaard et al. [20] study, Faujasite (FAU) and EMT were poor binding substrates for LPG (Fig. 3). Both have low Si/Al ratios. Under our binding assay conditions LPG displayed only 40–60% binding affinity to these zeolites. Similarly, two Linde Type A (LTA) zeolites (Valfor 100 and Molecular Sieve 5A) and one FAU-X (Molecular Sieve 13X) type of zeolite tested here showed LPG affinity comparable to that obtained with EMT and FAU (Fig. 3). LPG displayed no affinity to CBV400 (Faujasite-Y, FAU-Y); CP914C (Ferrierite, FER); CP814E (Beta Polymorph A, BEA) and CBV2314 (ZSM-5, MFI). However, our characterization has identified two Mordenite (CBV10A and CBV21A) and two Faujasite-Y www.elsevier.com/locate/nbt
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properties of the silica samples tested. LPG also displayed weak affinity to 3 different glass powders, which usually have a silica composition between 31 and 70% (Fig. 4). Beads and glass were ground down to obtain the final powder. Only 40% of the LPG was found in the bound fraction of glass beads (74% silica), whereas 20% was found in the bound fractions of zirconia/glass beads (31% silica) and flint glass powders (45% silica).
Effect of protein charge on binding Research Paper FIGURE 3
Binding affinity of purified LPG to commercial synthetic zeolites (5 mg each). Binding was performed as described in ‘Materials and methods’ section. Wash fractions (3 100 ml) were not loaded onto the gels. S, starting LPG (30 mg); U, unbound LPG fraction; B, zeolite-bound LPG fraction; LPG, Linker-Protein G.
(CBV100 and CBV300) types of zeolites as the best binding substrates for the purified LPG (Fig. 3). With these zeolites, 100% of the initial LPG was found in the final bound fraction.
Silica The affinity of LPG was tested against 12 different types of commercial silica substrates (Fig. 4). LPG displayed high binding affinity to all silica samples, except for Silica LC 60A (50% binding affinity) and Silica Gel 60 (30% binding affinity). LPG displayed between 90 and 100% binding affinity to all other commercial silica samples tested. Supplementary Table 2 summarizes some
Binding assays with LPG and the control proteins without linker sequence were performed with selected silica-containing materials to confirm that the binding observed was dependent on the linker sequence (Fig. 5). LPG displayed high binding affinity to all the zeolites and silicas tested (Fig. 5a). Protein G’ without the linker sequence (Fig. 5b) displayed no binding affinity towards FAU-Y synthetic zeolites (CVB100 and CVB 300). However, with MOR zeolites (CBV10A and CVB21A), approximately 40% of the Protein G’ remained bound. Protein G’ displayed no affinity towards the natural zeolite clinoptilolite and less than 5% remained bound to silica. By contrast, the basic recombinant Dictyoglomus XynB was expected to have a positive overall charge at the assay pH and to bind to the negatively charged zeolite and silica. However, under our binding assay conditions, Dictyoglomus XynB displayed no binding affinity toward any of the zeolite-containing materials tested (Fig. 5c). Binding assays with Dictyoglomus XynB and zeolite CBV100 at pH values between 4 and 9 were also performed to assess the effect of the overall protein charge on adsorption to zeolite. Under these conditions, Dictyoglomus XynB was unable to bind to the zeolite at any pH tested (Fig. 6).
Minimal number of repeats of the linker required for binding The actual sequence described in Nygaard et al. [20] for plasmid pSN6 encoded for a 21 amino acid residues peptide (VKTQATSREEPPRLPSKHRPG) repeated four times plus another 7 residues of
FIGURE 4
Binding affinity of purified LPG to silica and silica-containing materials (5 mg each). Binding was performed as described in ‘Materials and methods’ section. Wash fractions (3 100 ml) were not loaded onto the gels. S, starting LPG (30 mg); U, unbound LPG fraction; B, silica-bound LPG fraction; LPG, Linker-Protein G. 488
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FIGURE 5
Binding affinity of (a) purified LPG; (b) commercial Protein G’; and (c) basic Dictyoglomus XynB to commercial synthetic zeolite, silica and Castle Mountain natural clinoptilolite (106–250 mm fraction). (a) Binding was performed as described in ‘Materials and methods’ section. Each substrate tested was at a concentration of 5 mg. Wash fractions (3 100 ml) were not loaded onto the gels. S, starting protein (30 mg); U, unbound protein fraction; B, substrate-bound protein fraction; FAU, faujasite; MOR, mordenite; CLI, clinoptilolite; SIL, silica.
a partial 5th repeat. The fusion protein expressed, APpSN6 (AP, alkaline phosphatase; pSN6, linker peptide), was shown to bind to zeolite. However, there was no indication of the specific requirement for a sequence repeated four times. Binding assays with LPG (4 linker sequence repeats) and truncated derivatives indicated that the minimum number of repeats required for complete binding to zeolite or silica was 3 (Fig. 7). There was no clear difference in the binding efficiency toward both substrates with either 3 or 4 repeats. The truncated derivative with only 2 repeats was still able to bind to zeolite and silica but displayed less than half the affinity shown by the two other derivatives. There was no difference in the binding affinity
FIGURE 6
Binding affinity of purified basic Dictyoglomus XynB to synthetic zeolite CBV100. Binding was performed over pH range 4–9. Zeolite tested was at a concentration of 5 mg. Wash fractions (3 100 ml) were not loaded onto the gels. S, starting protein (30 mg); U, unbound protein fraction; B, zeolite-bound protein fraction.
between the derivative carrying one linker sequence and Protein G’ without linker sequence. In both cases only a non-specific binding of less than 5% was observed. Similarly GBP1, a 14 amino acid gold-binding peptide (MHGKTQATSGTIQS) required at least 3 tandem repeats for high-affinity binding to gold particles [17]. These results indicate that the binding avidity of LPG and GBP1 was directly dependent on the number of repeated sequences present.
Discussion The original report by Nygaard et al. [20] indicated that the zeolitespecific linker peptide displayed affinity to only two zeolites with high charge density (EMT and FAU, low Si/Al ratio), while the linker did not bind to the low charge density (high Si/Al ratio) AFI or MFI zeolites. According to these authors, neither the charge density nor the basic nature of the protein tested (net positive charge at assay pH) carrying the linker was responsible for the ability of the protein to bind to the zeolites. The main determinant for binding was claimed to be the ability of the linker peptide to recognise surface features unique to the face of the EMT zeolite. Generally zeolites are allowed to equilibrate to standard water content before conducting ion-exchange or adsorption studies. In the current study, however, we were interested in testing as many raw (without pre-conditioning) silica-containing materials as possible to determine better the substrate affinity of the linker. This approach was necessary also in order (a) to compare our results with the previous work of Nygaard et al. [20] and (b) to test the linker peptide suitability for later applications using untreated, commercially available natural and synthetic materials. We carried out a more comprehensive characterization of the linker peptide (in the form of the fusion protein LPG) with two natural zeolites and 13 synthetic zeolites. Under our binding assay conditions, purified LPG displayed only 40–60% binding affinity to the original low Si/Al zeolites (EMT and FAU) from Nygaard (both zeolite samples were kindly provided by S. Brown). Similar www.elsevier.com/locate/nbt
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FIGURE 7
Binding assay of truncated derivatives. Purified truncated LPG derivatives were incubated with 5 mg of zeolite CBV 100 (a) or precipitated silica (b) as described in Materials and Methods. Unbound and bound fractions were resolved by SDS-PAGE and visualised by staining with Coomassie Brilliant Blue. PG’, truncated Protein G without linker sequence; 1 LPG, truncated LPG carrying a single linker sequence; 2 LPG, truncated LPG carrying two linker sequence repeats; 3 LPG, truncated LPG carrying three linker sequence repeats; 4 LPG, LPG carrying four linker sequence repeats (LPG). S, starting protein (30 mg); U, unbound protein fraction; B, bound protein fraction; M, Novex sharp protein standard (Invitrogen). Wash (3 100 ml) fractions were not loaded onto the gels.
binding affinities were observed against two LTA zeolites with low Si/Al and one FAU-X zeolite with an intermediate Si/Al ratio. However, contrary to their previous findings, LPG was able to bind with higher affinity to low charge density mordenites (high Si/Al ratio). Thus, the linker peptide displayed affinity not only to low Si/Al (high charge density) zeolites but also to those with intermediate and high Si/Al ratios (intermediate and low charge density). Despite these observations it is difficult to assign binding trends purely based on the properties of the zeolites tested, their adsorption and ion-exchange properties. We suggest a similar linker-mediated binding mechanism to that observed with other peptide sequences exhibiting affinity for inorganic matrices (see discussion below). Natural zeolites, especially clinoptilolite, have a variety of applications mainly due to their abundance and ion-exchange properties. In the water and wastewater treatment industries, clinoptilolites have been introduced successfully for the removal of ammonium, the most common cation found in wastewaters [23–25]. They are used widely as a catalyst to separate and purify gases in the petrochemical industry and as a component of fertilizer in agriculture and as animal feed additives. Despite the extensive variety of applications for natural zeolite, to our knowledge, they have not been used as an immobilization matrix or carrier for proteins or other biomolecules. Furthermore, we are not aware that the binding affinity of the linker described here has been demonstrated previously with natural clinoptilolite zeolite. Accordingly, the results presented in this study clearly indicate that the affinity of the zeolite-specific linker peptide extends beyond the original claims of Nygaard et al. [20] and that unique surface features on the face of the EMT zeolite are not solely responsible for linker affinity. The silica materials tested in the experiments reported here were characterized by their high SiO2 content, which ranged between 82 and 99.4%. It was anticipated from the report of Nygaard et al. [20] that the affinity of the linker-peptide to EMT zeolite was a consequence of the polypeptide being able to recognise and 490
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distinguish subtle differences in the spatial orientation of the crystal surface atoms. If this were the case, we expected that the linker-peptide would have a very narrow and selective affinity for certain zeolites. However, the LPG affinity data presented here indicated that the linker affinity is much broader than originally claimed. Furthermore, the silica affinity data may imply that the binding affinity is directed to the SiO2 component (content and/or arrangement) rather than to the atomic orientation on zeolite crystal surface. This result is of especial interest because some of the silicas tested here have a composition of over 99.4% SiO2. Zeolite structures have pores in the size range of 0.3–3.0 nm, which are too small to allow entry of proteins. They cannot function as biomolecular sieves because of their small pore diameter. Thus, adsorption of proteins occurs only on the external surface of the crystals [26]. Although the molecular mass of the proteins has no relation to their adsorption efficiency, it has been shown that the extent of protein adsorption to zeolites is pHdependent and increases at or near their isolectric point (pI) [16,26–28]. Matsui et al. [28] studied the suitability of zeolites as a new form of carrier for chromatographic separation of proteins. They showed that adsorption of several proteins was zeolite species-specific and was dependent on the pH of the binding assay. Because the overall charge of zeolites is negative, the sum of attraction and repulsion electrostatic forces may influence adsorption. At pH values below the pI, proteins will carry an overall positive charge and adsorption will be driven mainly by Coulomb’s electrostatic attractions. When pH = pI, protein aggregates lose their electrical charge and adsorption is driven by hydrophobic interactions, surface structure and the number of mesopores (pore size between 2 and 50 nm). At pH’s above the pI, electrostatic repulsion between the negatively charged protein and zeolite may occur and thus absorption is driven by the sum of the Columbic repulsion and attractive forces (e.g., hydrophobic interactions). Protein G’ (lacking the linker) and LPG carry an overall negative charge while the basic Dictyoglomus XynB has an overall positive charge, based on the theoretical pI’s deduced from their amino
acid sequences at the pH of the assay. Zeolite and silica provide an extensive negatively charged surface for the interaction of positively charged molecules. Our results show that the presence of the zeolite-specific linker peptide mediates the binding between the protein and the silica-containing materials. The basic protein, XynB, lacking the linker peptide was unable to bind to zeolite even when the binding assay was performed over a wide pH range (4–9). XynB carries a very high overall positive charge at the lowest pH of the binding assay. Similarly, a Protein G’ lacking the linker sequence also displayed no affinity to the negatively charged zeolite or silica substrates. Taniguchi et al. [4] characterized a silica-binding tag (Si-tag) derived from E. coli ribosomal protein L2. The N- and C-terminal regions of the Si-tag contained 63 positively charged amino acid residues, conferring on this peptide a theoretical isoelectric point of 10.9. Binding of Si-tag to silica was pH-dependent, with maximum binding reached at pH 8.0. At pH values above 7.0, Si-tag was positively charged while silica displayed a net negative charge. Sarikaya et al. [29] reviewed 28 different polypeptide sequences exhibiting affinity for various inorganics (e.g., Au, SiO2, zeolites, ZnO, and so on). Over 70% of the polypeptides were positively charged and carried a large proportion of basic amino acid residues. This high net charge seems to be a property of polypeptides with high affinity to inorganics and might reflect tendencies toward amino acid residues that promote a less stable ordered structure (see below). Theoretical predictions of disorder regions in L2 protein (Si-tag) have shown that the two highly charged regions (N- and Cterminal regions) represented areas of high disorder while the central domain of L2 represented a highly ordered region [30]. These results and previous attempts to obtain the complete crystal structure of L2 with these regions [31,32] further support the lack of a defined three-dimensional structure at both ends of L2. Over 76% of the amino acid residues of the linker peptide of LPG presented here represent residues with tendency to promote
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structural disorder [33]. These residues confer low overall hydrophobicity and high net charge, a characteristic property of polypeptide sequences exhibiting affinity to inorganic compounds [29]. The intrinsic structural disorder of these peptides seems to play an important role in the binding to inorganics. The positively charged residues in the disordered region of the linker form multiple ionic interactions with the negatively charged silica or zeolite surface. The strong binding affinity of the linker peptide to these silica-containing materials can be attributed to the flexibility and plasticity of the peptide being able to adopt an optimal conformation for binding substrates with differently shaped topologies or surfaces. These types of linkers are already in an unfolded state and adsorption and conformational adaptation to the substrate (e.g., silica) binding interface are a fast and concomitant events [30].
Conclusion The linker peptide sequence presented here displayed high affinity towards silica-containing materials. Thus, the linker as part of a fusion protein in conduction with natural and synthetic zeolites or silica can be used for purification or immobilization of proteins. These materials not only are less expensive than conventional protein purification materials (e.g., agarose and sepharose) but also are more stable to high temperatures, pressure or extreme pH’s.
Acknowledgements The authors are grateful to Zeolite Australia Pty Ltd. and Castle Mountain Zeolites Pty Ltd. for suppling natural zeolite samples. We thank Dr. M. Gibbs for providing samples of purified Dictyoglomus XynB enzyme. This work was funded by the Environmental Biotechnology Cooperative Research Centre (EBCRC).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.nbt.2012.11.022.
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[13] Camarero JA. Recent developments in the site-specific immobilization of proteins onto solid supports. Peptide Science 2008;90:450–8. [14] Breck DW. Zeolite molecular sieves: structure chemistry and use. John Wiley; 1974. [15] Szostak R. Molecular sieves: principles of synthesis and identification. Van Nostrand Reinhold; 1989. [16] Tavolaro A, Tavolaro P, Drioli E. Influence of synthesis parameters on vanadiumsilicalite-1 crystal growth prepared with fluoride-containing media. Journal of Crystal Growth 2006;289:609–16. [17] Brown S. Metal-recognition by repeating polypeptides. Nature Biotechnology 1997;15:269–72. [18] Brown S. Protein-mediated particle assembly. Nano Letters 2001;1:391–4. [19] Brown S, Sarikaya M, Johnson E. A genetic analysis of crystal growth. Journal of Molecular Biology 2000;299:725–35. [20] Nygaard S, Wendelbo R, Brown S. Surface-specific zeolite-binding proteins. Advanced Materials 2002;14:1853–6. [21] Goward CR, Murphy JP, Atkinson T, Barstow DA. Expression and purification of a truncated recombinant streptococcal protein G. Biochemical Journal 1990;267:171–7. [22] Morris DD, Gibbs MD, Chin CW, Koh MH, Wong KK, Allison RW, et al. Cloning of the xynB gene from Dictyoglomus thermophilum Rt46B.1 and action of the gene product on kraft pulp. Applied and Environmental Microbiology 1998;64:1759–65. [23] Nguyen ML, Tanner CC. Ammonium removal from wastewaters using natural New Zealand zeolites. New Zealand Journal of Agricultural Research 1998;41:427–46. ¨ zdemir B, Turan M, Koyuncu I, Atesok G, Sarikaya HZ. Removal of [24] C ¸ elik MS, O ammonia by natural clay minerals using fixed and fluidised bed column reactors. Water Science and Technology Water Supply 2001;1:81–8. [25] Jorgensen TC, Weatherley LR. Ammonia removal from wastewater by ion exchange in the presence of organic contaminants. Water Research 2003;37:1723–8.
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[26] Klint D, Arvidsson P, Blum Z, Eriksson H. Purification of proteins by the use of hydrophobic zeolite-Y. Protein Expression and Purification 1994;5: 569–76. [27] Klint D, Eriksson H. Conditions for the adsorption of proteins on ultrastable zeolite Y and its use in protein purification. Protein Expression and Purification 1997;10:247–55. [28] Matsui M, Kiyozumi Y, Yamamoto T, Mizushina Y, Mizukami F, Sakaguchi K. Selective adsorption of biopolymers on zeolites. Chemistry – A European Journal 2001;7:1555–60. [29] Sarikaya M, Tamerler C, Jen AK, Schulten K, Baneyx F. Molecular biomimetics: nanotechnology through biology. Nature Materials 2003;2:577–85. [30] Ikeda T, Kuroda A. Why does the silica-binding protein Si-tag bind strongly to silica surfaces? Implications of conformational adaptation of the intrinsically
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disordered polypeptide to solid surfaces. Colloids Surfaces B Biointerfaces 2011;86:359–63. [31] Nakashima T, Kimura M, Nakagawa A, Tanaka I. Crystallization and preliminary X-ray crystallographic study of a 23S rRNA binding domain of the ribosomal protein L2 from Bacillus stearothermophilus. Journal of Structural Biology 1998;124:99–101. [32] Nakagawa A, Nakashima T, Taniguchi M, Hosaka H, Kimura M, Tanaka I. The three-dimensional structure of the RNA-binding domain of ribosomal protein L2; a protein at the peptidyl transferase center of the ribosome. EMBO Journal 1999;18:1459–67. [33] Campen A, Williams RM, Brown CJ, Meng J, Uversky VN, Dunker AK. TOP-IDPscale: a new amino acid scale measuring propensity for intrinsic disorder. Protein & Peptide Letters 2008;15:956–63.
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Research Paper
A linker peptide with high affinity towards silica-containing materials Anwar Sunna1, Fei Chi1 and Peter L. Bergquist1,2 1 2
Department of Chemistry and Biomolecular Sciences, and Environmental Biotechnology CRC, Macquarie University, Sydney, Australia Department of Molecular Medicine & Pathology, University of Auckland Medical School, Auckland, New Zealand
A peptide sequence with affinity to silica-containing materials was fused to a truncated form of Streptococcus strain G148 Protein G. The resulting recombinant Linker-Protein G (LPG) was produced in Escherichia coli and purified to apparent homogeneity. It displayed high affinity towards two natural clinoptilolite zeolites. The LPG also displayed high binding affinity towards commercial-grade synthetic zeolite, silica and silica-containing materials. A commercial sample of the truncated Protein G and a basic protein, both without the linker, did not bind to natural or synthetic zeolites or silica. We conclude that the zeolite-binding affinity is mediated by the linker peptide sequence. As a consequence, these data may imply that the binding affinity is directed to the SiO2 component rather than to the atomic orientation on the zeolite crystal surface as previously assumed.
Introduction The interaction of proteins with solid surfaces is central to several chromatographic purification and biosensor techniques [1]. The application of unmodified silica in protein purification has been used relatively rarely [2]. More common are methods in which a biological recognition molecule is immobilized onto a silica or silica-based sensing substrate. Methods involving covalent immobilization are generally achieved in three steps: modification of the surface to add specific functional groups, covalent attachment of a crosslinker through one of its reactive moieties and covalent linking of the biomolecular recognition element to the remaining reactive sites of the crosslinker. Methods have been described to allow immobilization of biological recognition molecules onto sensing surfaces with full functionality in biosensor-binding assays. A frequent theme has been the orientated immobilization of antibodies on silicon wafers [3,4] or nanowires as electronic devices for sensitive and direct detection of biological analytes [5]. Significant progress has been made on a range of immobilization supports [6,7]. However, the immobilization of proteins has been a particularly challenging task, mainly due to their heterogeneous nature and the marginal stability of the active tertiary Corresponding author: Sunna, A. ([email protected])
structure over the denatured (and inactive) random coil structure. Most of the methods available for immobilizing proteins onto solid supports traditionally have relied on nonspecific adsorption [8,9] or on the reaction of naturally occurring chemical groups such as amines and carboxylic acids within proteins with appropriate reactive groups on the matrix [10,11]. In both cases, the corresponding proteins are attached to the surface in a random orientation that may cause the reduction or loss of the protein’s biological activity [12]. Recent reviews have summarized progress and the status of the field [7,13]. The techniques described above are elegant but may not lend themselves to inexpensive bulk applications particularly suited to field conditions. Zeolites are inorganic materials that have a highly ordered structure and can be synthesized as nanocrystals with pores and channel systems in the molecular size range of 0.3–3.0 nm. They offer interesting characteristics as inorganic support materials to immobilize proteins including mechanical and chemical resistance and high surface area [14,15]. The basic/acidic nature of zeolites can be modified by varying the Si/Al ratio or by introducing different metals into the crystalline framework and by changing the Si/metal ratio [16]. The acidity of zeolites can be modified by exchanging extra-framework metal cations with H+ ions. They are known to be stable both in wet and dry conditions.
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Brown [17–19] reported that repeating polypeptides fused to protein sequences could bind to their target metals with high affinity and specificity. Nygaard et al. [20] selected specific zeolitebinding proteins by Escherichia coli cell-surface display. The candidate sequences for zeolite-binding proteins were transferred from the gene encoding the bacterial surface protein (lamB) and fused to the alkaline phosphatase (phoA) gene. The soluble fusion protein expressed was shown to bind to zeolite and displayed enzymatic activity. One sequence described for plasmid pSN6 coded for 21 amino acid residues (VKTQATSREEPPRLPSKHRPG) repeated four times plus another 7 residues of a partial 5th repeat [20]. The fusion protein was reported to bind specifically only to two synthetic zeolites, Faujasite (FAU) and EMT (both synthesized by SINTEF, Norway) but not to other zeolites. We reasoned that other materials containing silica might be bound by the same or a related linker sequence that permits specific binding to zeolite. We fused the above linker to a truncated form of Streptococcus Protein G, Protein G’, [21] and examined the ability of the resulting recombinant protein to bind to silica-containing materials. In this work we present a detailed and extensive examination of the binding affinity of this recombinant protein.
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Production and purification of recombinant LPG
CBV and CP zeolite series were purchased from Zeolyst International (Conshohocken, PA, USA), Valfor 100 zeolite was provided by PQ Australia Pty Ltd. (Dandenong South, VIC, Australia), molecular sieves 5A and 13X were from Fujian Anten Chemical Co. Ltd. (Xiamen, China), clinoptilolite samples were provided by Zeolite Australia Pty Ltd. (Werris Creek, NSW, Australia) and Castle Mountain Zeolites Pty Ltd. (Quirindi, NSW, Australia). Precipitated silica was from BDH Ltd., Davisil silica grade LC 60A and 646, silica Perkasil, Elfadent and Durafill were supplied by Grace Davison Discovery Sciences (Rowville, VIC, Australia), Scharlau silica gel 60 was obtained from Chem-Supply Pty Ltd. (Adelaide, SA, Australia), silica Rhodoline and Tixosil from Rhodia were provided by Brenntag Pty Ltd. (Notting Hill, VIC, Australia).
For the production of recombinant LPG, 300 ml Luria Bertani (LB) medium supplemented with 50 mg/ml carbenicillin was inoculated with 3 ml of an overnight culture of E. coli Tuner (DE3) cells (Novagen) harbouring pOPT_LPGpET22b. The culture was incubated at 37 8C with shaking (250 rpm) until the A600 was approximately 0.7–1.0. The incubation temperature was reduced to 20 8C and protein synthesis was induced by the addition of 0.2 mM isopropyl b-D-thiogalactoside (IPTG). Cells were harvested after 3– 4 h induction by centrifugation for 15 min at 10,000 g and 4 8C and were stored at 20 8C. The cells were resuspended in ice-cold lysis buffer (25 mM Tris– HCl, pH 8.0, 100 mM NaCl, 1.25 mM EDTA and 0.05% Tween 20), supplemented with 4 mM of the Pefabloc serine protease inhibitor (Roche) and 1.5 mg lysozyme (Sigma). They were ruptured by three passages through a French pressure cell. After cell rupture, 50 U of DNase I (Invitrogen) and 4 mM Pefabloc were added. The sample was incubated with rotation at room temperature for 20 min. The debris was removed by centrifugation for 30 min at 20,000 g and 4 8C. The supernatant obtained was first filtered through a 0.45 mm and then through a 0.2 mm sterile filter and stored at 4 8C. The extract was loaded onto a 5 ml HiTrap Q anion exchanger column (GE Healthcare) previously equilibrated with 25 mM Tris– HCl, pH 8.0, supplemented with 100 mM NaCl. The column was washed extensively with the same buffer. Under these conditions the LPG was found in the 100 mM NaCl fraction. The LPG fraction was applied to a 5 ml HiTrap SP cation exchanger column (GE Healthcare) previously equilibrated with buffer containing 100 mM NaCl. The column was washed extensively with the same buffer and the LPG was eluted at 150 and 200 mM NaCl. Fractions containing the LPG were identified on a NuPAGE 4–12% Bis–Tris Gel (1.5 mm, 10 well, Invitrogen) by SDS-PAGE and staining with Coomassie Brilliant Blue and pooled and concentrated using an Amicon Ultra-15 centrifugal filter (10 kDa cut-off, Millipore). Samples were stored in 25 mM Tris–HCl, pH 8.0, 100 mM NaCl at 20 8C after sterile filtration through a 0.1 mm filter.
Construction of a Linker-Protein G (LPG) recombinant protein
Minimal number of repeats required for binding
The Protein G sequence used was based on that reported by Goward et al. [21] which codes for a truncated recombinant form of Streptococcus strain G148 Protein G (Protein G’). A DNA sequence encoding the Linker amino acid sequence (VKTQATSREEPPRLPSKHRPG)4VKTQTAS and a DNA sequence encoding a truncated Protein G’ DNA sequence were joined with the linker sequence positioned at the N-terminus of Protein G’. The codon usage of the final combined Linker and Protein G’ sequence was optimized for expression in E. coli. Rare codons and negative cisacting motifs (such as internal RBS sites, TATA-boxes, Chi-recombination sites, and so on) that can hamper expression in E. coli were removed during codon optimization. Codon optimization and gene synthesis of the final combined sequence was performed by GENEART AG (Regensburg, Germany). The final optimized sequence (OPT_LPG) was designed with flanking restriction sites (NdeI and BamHI) for ligation into plasmid pET22b (Novagen) to give the expression plasmid pOPT_LPGpET22b. The protein expressed from the codon-optimized OPT_LPG sequence is referred to here as Linker-Protein G (LPG).
Several truncated derivatives of pOPT_LPGpET22b (Fig. 1a) were prepared to determine the minimum number of repeat sequences required for binding of LPG to silica-containing materials. DNA fragments encoding the different truncated recombinant proteins were amplified by PCR using the primer pairs described in Fig. 1b. The amplified DNA fragments were digested with NdeI/BamHI and ligated into similarly cut pET22b. Production and purification of the different truncated recombinant LPG proteins was as described above.
Materials and methods Zeolite and silica
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Standard binding assay The binding of purified recombinant LPG to silica-containing material (e.g., natural and synthetic zeolite and silica) was determined as follows. Silica-containing materials were washed three times with washing buffer (10 mM Tris–HCl, pH 7.5, 100 mM NaCl and 1% Triton-X100). Soluble protein in a final volume of 100 ml was mixed with silica-containing material and incubated by rotation at room temperature for 1 h. The unbound fraction was removed after centrifugation at 14,000 g for 20 s. The pellet of
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FIGURE 1
Truncated derivatives used in this study. (a) Plasmids, primers and architecture for each truncated derivative. (b) Oligonucleotides used in this study. aNumber of linker sequence repeats; bN.A, not applicable; cpurified recombinant truncated Protein G’ (Sigma–Aldrich); dengineered restriction sites are underlined.
silica-containing material was washed three times by vortexing with 100 ml of 100 mM Tris–HCl buffer, pH 8.0. The bound protein was eluted after addition of 100 ml of SDS PAGE-loading buffer and incubation at 99 8C for 10 min (with short mixing every 2 min). Fractions containing the protein were identified by SDS-PAGE and staining with Coomassie Brilliant Blue. In addition, a control-binding assay was performed with LPG and two other proteins lacking the linker sequence. Truncated Protein G’ (without the linker, Sigma–Aldrich) and a basic Dictyoglomus XynB xylanase [22] were used as controls for the binding experiments. Binding assays with Dictyoglomus XynB at different pH values were performed to assess the effect of the overall protein charge on adsorption to silica-containing materials. Dictyoglomus XynB, binding and washing buffers were adjusted to the final pH of the experiment. The following buffers at a final concentration of 100 mM each were used in the assay: Na-acetate buffer (pH 4 and 5); 2-(N-Morpholino) ethanesulfonic acid buffer (MES, pH 6); Tris buffer (pH 7–9). The percentage of protein in the different fractions was semiquantified by analysing the intensity of bands of digital images from SDS-PAGE with ImageJ software (http://rsb.info.nih.gov/ij/ index.html).
Results Purification of recombinant LPG The expression plasmid pOPT_LPGpET22b was introduced into E. coli Tuner (DE3) cells and recombinant protein production and purification were carried out as described in the ‘Materials and methods’ section. The optimized recombinant LPG protein from expression in E. coli was purified to electrophoretic homogeneity as evaluated by SDS-PAGE (data not shown). The final yield of pure LPG protein from 300 ml of medium was approximately 20 mg.
Affinity of LPG towards silica-containing materials Natural zeolite Two types of natural zeolites were tested (Fig. 2). Zeolite Australia is a zeolite comprising a 54% clinoptilolite content while Castle
FIGURE 2
Binding affinity of purified LPG to Castle Mountain and Zeolite Australia natural clinoptilolite (5 mg each). Binding was performed as described in ‘Materials and methods’ section. Wash fractions (3 100 ml) were not loaded onto the gels. S, starting LPG (80 mg); U, unbound LPG fraction; B, zeolitebound LPG fraction; LPG, Linker-Protein G.
Mountain zeolite contains 85% clinoptilolite. The cationexchange capacity (CEC) for Zeolite Australia and Castle Mountain zeolites are reported as 1.19 and 1.47 meq/g, respectively (http:// www.zeolitemineral.com/worldwide.php, March 2012). The Zeolite Australia zeolite (7 Mohs) is much harder than the Castle Mountain zeolite (5 Mohs). The purified LPG showed affinity to both types of natural zeolites (particle size <10 mm to 2.0 mm). Most of the LPG was found in the bound fraction when incubated with zeolites with particle size <10 mm to 0.5 mm. However, this affinity decreased with zeolite particles above 0.5 mm. This might be a result of larger particles having less surface area available for LPG binding. The differences in the degree of binding due to the particle size could be overcome by increasing the amount of zeolite used in the experiments (data not shown). This finding probably supports the observation that the differences are a consequence of the reduced surface area availability in larger zeolite particles.
Synthetic zeolite Thirteen synthetic zeolites belonging to 8 different zeolite families were tested as binding substrates for the purified LPG. Supplementary Table 1 summarizes some properties of the synthetic zeolites tested. This comprehensive characterization of the linker peptide (LPG) showed that the two zeolites originally reported in the Nygaard et al. [20] study, Faujasite (FAU) and EMT were poor binding substrates for LPG (Fig. 3). Both have low Si/Al ratios. Under our binding assay conditions LPG displayed only 40–60% binding affinity to these zeolites. Similarly, two Linde Type A (LTA) zeolites (Valfor 100 and Molecular Sieve 5A) and one FAU-X (Molecular Sieve 13X) type of zeolite tested here showed LPG affinity comparable to that obtained with EMT and FAU (Fig. 3). LPG displayed no affinity to CBV400 (Faujasite-Y, FAU-Y); CP914C (Ferrierite, FER); CP814E (Beta Polymorph A, BEA) and CBV2314 (ZSM-5, MFI). However, our characterization has identified two Mordenite (CBV10A and CBV21A) and two Faujasite-Y www.elsevier.com/locate/nbt
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properties of the silica samples tested. LPG also displayed weak affinity to 3 different glass powders, which usually have a silica composition between 31 and 70% (Fig. 4). Beads and glass were ground down to obtain the final powder. Only 40% of the LPG was found in the bound fraction of glass beads (74% silica), whereas 20% was found in the bound fractions of zirconia/glass beads (31% silica) and flint glass powders (45% silica).
Effect of protein charge on binding Research Paper FIGURE 3
Binding affinity of purified LPG to commercial synthetic zeolites (5 mg each). Binding was performed as described in ‘Materials and methods’ section. Wash fractions (3 100 ml) were not loaded onto the gels. S, starting LPG (30 mg); U, unbound LPG fraction; B, zeolite-bound LPG fraction; LPG, Linker-Protein G.
(CBV100 and CBV300) types of zeolites as the best binding substrates for the purified LPG (Fig. 3). With these zeolites, 100% of the initial LPG was found in the final bound fraction.
Silica The affinity of LPG was tested against 12 different types of commercial silica substrates (Fig. 4). LPG displayed high binding affinity to all silica samples, except for Silica LC 60A (50% binding affinity) and Silica Gel 60 (30% binding affinity). LPG displayed between 90 and 100% binding affinity to all other commercial silica samples tested. Supplementary Table 2 summarizes some
Binding assays with LPG and the control proteins without linker sequence were performed with selected silica-containing materials to confirm that the binding observed was dependent on the linker sequence (Fig. 5). LPG displayed high binding affinity to all the zeolites and silicas tested (Fig. 5a). Protein G’ without the linker sequence (Fig. 5b) displayed no binding affinity towards FAU-Y synthetic zeolites (CVB100 and CVB 300). However, with MOR zeolites (CBV10A and CVB21A), approximately 40% of the Protein G’ remained bound. Protein G’ displayed no affinity towards the natural zeolite clinoptilolite and less than 5% remained bound to silica. By contrast, the basic recombinant Dictyoglomus XynB was expected to have a positive overall charge at the assay pH and to bind to the negatively charged zeolite and silica. However, under our binding assay conditions, Dictyoglomus XynB displayed no binding affinity toward any of the zeolite-containing materials tested (Fig. 5c). Binding assays with Dictyoglomus XynB and zeolite CBV100 at pH values between 4 and 9 were also performed to assess the effect of the overall protein charge on adsorption to zeolite. Under these conditions, Dictyoglomus XynB was unable to bind to the zeolite at any pH tested (Fig. 6).
Minimal number of repeats of the linker required for binding The actual sequence described in Nygaard et al. [20] for plasmid pSN6 encoded for a 21 amino acid residues peptide (VKTQATSREEPPRLPSKHRPG) repeated four times plus another 7 residues of
FIGURE 4
Binding affinity of purified LPG to silica and silica-containing materials (5 mg each). Binding was performed as described in ‘Materials and methods’ section. Wash fractions (3 100 ml) were not loaded onto the gels. S, starting LPG (30 mg); U, unbound LPG fraction; B, silica-bound LPG fraction; LPG, Linker-Protein G. 488
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FIGURE 5
Binding affinity of (a) purified LPG; (b) commercial Protein G’; and (c) basic Dictyoglomus XynB to commercial synthetic zeolite, silica and Castle Mountain natural clinoptilolite (106–250 mm fraction). (a) Binding was performed as described in ‘Materials and methods’ section. Each substrate tested was at a concentration of 5 mg. Wash fractions (3 100 ml) were not loaded onto the gels. S, starting protein (30 mg); U, unbound protein fraction; B, substrate-bound protein fraction; FAU, faujasite; MOR, mordenite; CLI, clinoptilolite; SIL, silica.
a partial 5th repeat. The fusion protein expressed, APpSN6 (AP, alkaline phosphatase; pSN6, linker peptide), was shown to bind to zeolite. However, there was no indication of the specific requirement for a sequence repeated four times. Binding assays with LPG (4 linker sequence repeats) and truncated derivatives indicated that the minimum number of repeats required for complete binding to zeolite or silica was 3 (Fig. 7). There was no clear difference in the binding efficiency toward both substrates with either 3 or 4 repeats. The truncated derivative with only 2 repeats was still able to bind to zeolite and silica but displayed less than half the affinity shown by the two other derivatives. There was no difference in the binding affinity
FIGURE 6
Binding affinity of purified basic Dictyoglomus XynB to synthetic zeolite CBV100. Binding was performed over pH range 4–9. Zeolite tested was at a concentration of 5 mg. Wash fractions (3 100 ml) were not loaded onto the gels. S, starting protein (30 mg); U, unbound protein fraction; B, zeolite-bound protein fraction.
between the derivative carrying one linker sequence and Protein G’ without linker sequence. In both cases only a non-specific binding of less than 5% was observed. Similarly GBP1, a 14 amino acid gold-binding peptide (MHGKTQATSGTIQS) required at least 3 tandem repeats for high-affinity binding to gold particles [17]. These results indicate that the binding avidity of LPG and GBP1 was directly dependent on the number of repeated sequences present.
Discussion The original report by Nygaard et al. [20] indicated that the zeolitespecific linker peptide displayed affinity to only two zeolites with high charge density (EMT and FAU, low Si/Al ratio), while the linker did not bind to the low charge density (high Si/Al ratio) AFI or MFI zeolites. According to these authors, neither the charge density nor the basic nature of the protein tested (net positive charge at assay pH) carrying the linker was responsible for the ability of the protein to bind to the zeolites. The main determinant for binding was claimed to be the ability of the linker peptide to recognise surface features unique to the face of the EMT zeolite. Generally zeolites are allowed to equilibrate to standard water content before conducting ion-exchange or adsorption studies. In the current study, however, we were interested in testing as many raw (without pre-conditioning) silica-containing materials as possible to determine better the substrate affinity of the linker. This approach was necessary also in order (a) to compare our results with the previous work of Nygaard et al. [20] and (b) to test the linker peptide suitability for later applications using untreated, commercially available natural and synthetic materials. We carried out a more comprehensive characterization of the linker peptide (in the form of the fusion protein LPG) with two natural zeolites and 13 synthetic zeolites. Under our binding assay conditions, purified LPG displayed only 40–60% binding affinity to the original low Si/Al zeolites (EMT and FAU) from Nygaard (both zeolite samples were kindly provided by S. Brown). Similar www.elsevier.com/locate/nbt
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FIGURE 7
Binding assay of truncated derivatives. Purified truncated LPG derivatives were incubated with 5 mg of zeolite CBV 100 (a) or precipitated silica (b) as described in Materials and Methods. Unbound and bound fractions were resolved by SDS-PAGE and visualised by staining with Coomassie Brilliant Blue. PG’, truncated Protein G without linker sequence; 1 LPG, truncated LPG carrying a single linker sequence; 2 LPG, truncated LPG carrying two linker sequence repeats; 3 LPG, truncated LPG carrying three linker sequence repeats; 4 LPG, LPG carrying four linker sequence repeats (LPG). S, starting protein (30 mg); U, unbound protein fraction; B, bound protein fraction; M, Novex sharp protein standard (Invitrogen). Wash (3 100 ml) fractions were not loaded onto the gels.
binding affinities were observed against two LTA zeolites with low Si/Al and one FAU-X zeolite with an intermediate Si/Al ratio. However, contrary to their previous findings, LPG was able to bind with higher affinity to low charge density mordenites (high Si/Al ratio). Thus, the linker peptide displayed affinity not only to low Si/Al (high charge density) zeolites but also to those with intermediate and high Si/Al ratios (intermediate and low charge density). Despite these observations it is difficult to assign binding trends purely based on the properties of the zeolites tested, their adsorption and ion-exchange properties. We suggest a similar linker-mediated binding mechanism to that observed with other peptide sequences exhibiting affinity for inorganic matrices (see discussion below). Natural zeolites, especially clinoptilolite, have a variety of applications mainly due to their abundance and ion-exchange properties. In the water and wastewater treatment industries, clinoptilolites have been introduced successfully for the removal of ammonium, the most common cation found in wastewaters [23–25]. They are used widely as a catalyst to separate and purify gases in the petrochemical industry and as a component of fertilizer in agriculture and as animal feed additives. Despite the extensive variety of applications for natural zeolite, to our knowledge, they have not been used as an immobilization matrix or carrier for proteins or other biomolecules. Furthermore, we are not aware that the binding affinity of the linker described here has been demonstrated previously with natural clinoptilolite zeolite. Accordingly, the results presented in this study clearly indicate that the affinity of the zeolite-specific linker peptide extends beyond the original claims of Nygaard et al. [20] and that unique surface features on the face of the EMT zeolite are not solely responsible for linker affinity. The silica materials tested in the experiments reported here were characterized by their high SiO2 content, which ranged between 82 and 99.4%. It was anticipated from the report of Nygaard et al. [20] that the affinity of the linker-peptide to EMT zeolite was a consequence of the polypeptide being able to recognise and 490
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distinguish subtle differences in the spatial orientation of the crystal surface atoms. If this were the case, we expected that the linker-peptide would have a very narrow and selective affinity for certain zeolites. However, the LPG affinity data presented here indicated that the linker affinity is much broader than originally claimed. Furthermore, the silica affinity data may imply that the binding affinity is directed to the SiO2 component (content and/or arrangement) rather than to the atomic orientation on zeolite crystal surface. This result is of especial interest because some of the silicas tested here have a composition of over 99.4% SiO2. Zeolite structures have pores in the size range of 0.3–3.0 nm, which are too small to allow entry of proteins. They cannot function as biomolecular sieves because of their small pore diameter. Thus, adsorption of proteins occurs only on the external surface of the crystals [26]. Although the molecular mass of the proteins has no relation to their adsorption efficiency, it has been shown that the extent of protein adsorption to zeolites is pHdependent and increases at or near their isolectric point (pI) [16,26–28]. Matsui et al. [28] studied the suitability of zeolites as a new form of carrier for chromatographic separation of proteins. They showed that adsorption of several proteins was zeolite species-specific and was dependent on the pH of the binding assay. Because the overall charge of zeolites is negative, the sum of attraction and repulsion electrostatic forces may influence adsorption. At pH values below the pI, proteins will carry an overall positive charge and adsorption will be driven mainly by Coulomb’s electrostatic attractions. When pH = pI, protein aggregates lose their electrical charge and adsorption is driven by hydrophobic interactions, surface structure and the number of mesopores (pore size between 2 and 50 nm). At pH’s above the pI, electrostatic repulsion between the negatively charged protein and zeolite may occur and thus absorption is driven by the sum of the Columbic repulsion and attractive forces (e.g., hydrophobic interactions). Protein G’ (lacking the linker) and LPG carry an overall negative charge while the basic Dictyoglomus XynB has an overall positive charge, based on the theoretical pI’s deduced from their amino
acid sequences at the pH of the assay. Zeolite and silica provide an extensive negatively charged surface for the interaction of positively charged molecules. Our results show that the presence of the zeolite-specific linker peptide mediates the binding between the protein and the silica-containing materials. The basic protein, XynB, lacking the linker peptide was unable to bind to zeolite even when the binding assay was performed over a wide pH range (4–9). XynB carries a very high overall positive charge at the lowest pH of the binding assay. Similarly, a Protein G’ lacking the linker sequence also displayed no affinity to the negatively charged zeolite or silica substrates. Taniguchi et al. [4] characterized a silica-binding tag (Si-tag) derived from E. coli ribosomal protein L2. The N- and C-terminal regions of the Si-tag contained 63 positively charged amino acid residues, conferring on this peptide a theoretical isoelectric point of 10.9. Binding of Si-tag to silica was pH-dependent, with maximum binding reached at pH 8.0. At pH values above 7.0, Si-tag was positively charged while silica displayed a net negative charge. Sarikaya et al. [29] reviewed 28 different polypeptide sequences exhibiting affinity for various inorganics (e.g., Au, SiO2, zeolites, ZnO, and so on). Over 70% of the polypeptides were positively charged and carried a large proportion of basic amino acid residues. This high net charge seems to be a property of polypeptides with high affinity to inorganics and might reflect tendencies toward amino acid residues that promote a less stable ordered structure (see below). Theoretical predictions of disorder regions in L2 protein (Si-tag) have shown that the two highly charged regions (N- and Cterminal regions) represented areas of high disorder while the central domain of L2 represented a highly ordered region [30]. These results and previous attempts to obtain the complete crystal structure of L2 with these regions [31,32] further support the lack of a defined three-dimensional structure at both ends of L2. Over 76% of the amino acid residues of the linker peptide of LPG presented here represent residues with tendency to promote
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structural disorder [33]. These residues confer low overall hydrophobicity and high net charge, a characteristic property of polypeptide sequences exhibiting affinity to inorganic compounds [29]. The intrinsic structural disorder of these peptides seems to play an important role in the binding to inorganics. The positively charged residues in the disordered region of the linker form multiple ionic interactions with the negatively charged silica or zeolite surface. The strong binding affinity of the linker peptide to these silica-containing materials can be attributed to the flexibility and plasticity of the peptide being able to adopt an optimal conformation for binding substrates with differently shaped topologies or surfaces. These types of linkers are already in an unfolded state and adsorption and conformational adaptation to the substrate (e.g., silica) binding interface are a fast and concomitant events [30].
Conclusion The linker peptide sequence presented here displayed high affinity towards silica-containing materials. Thus, the linker as part of a fusion protein in conduction with natural and synthetic zeolites or silica can be used for purification or immobilization of proteins. These materials not only are less expensive than conventional protein purification materials (e.g., agarose and sepharose) but also are more stable to high temperatures, pressure or extreme pH’s.
Acknowledgements The authors are grateful to Zeolite Australia Pty Ltd. and Castle Mountain Zeolites Pty Ltd. for suppling natural zeolite samples. We thank Dr. M. Gibbs for providing samples of purified Dictyoglomus XynB enzyme. This work was funded by the Environmental Biotechnology Cooperative Research Centre (EBCRC).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.nbt.2012.11.022.
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