One-Pot Glyco-Affinity Precipitation Purification for Enhanced Proteomics: The Flexible Alignment of Solution-Phase Capture/ Release and Solid-Phase Separation Xue-Long Sun,*,† Carolyn A. Haller†, XiaoYi Wu†, Vincent P. Conticello,‡ and Elliot L. Chaikof*,†,§ Departments of Surgery and Biomedical Engineering, Emory University School of Medicine and Georgia Institute of Technology, Atlanta, Georgia 30322, Department of Chemistry, Emory University, Atlanta, Georgia 30322, and School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332 Received July 28, 2005
Abstract: A one-pot affinity precipitation purification of carbohydrate-binding protein was demonstrated by designing thermally responsive glyco-polypeptide polymers, which were synthesized by selective coupling of pendant carbohydrate groups to a recombinant elastin-like triblock protein copolymer (ELP). The thermally driven inverse transition temperature of the ELP-based triblock polymer is maintained upon incorporation of carbohydrate ligands, which was confirmed by differential scanning calorimetry and 1H NMR spectroscopy experiments. As a test system, lactose derivatized ELP was used to selectively purify a galactose-specific binding lectin through simple temperature-triggered precipitation in a high level of efficiency. Potential opportunities might be provided for enhanced proteomic, cell isolation as well as pathogen detection applications. Keywords: glyco-affinity precipitation • protein purification • glycoconjugate • elastin-like polypeptide
Introduction Proteins are ultimately responsible for the biological processes in cells, body fluids, and tissue specimens. This presents enormous challenges to the field of proteomics, which aims to identify, characterize, and assign biological functions of all proteins. Determining individual protein in complex biological samples often requires some type of separation as a prerequisite for its measurement. The complexities of chemical structure and in the physiological function of every protein contribute to the problems encountered when trying to separate these biomolecules. A number of techniques exist for the separation of proteins, however, a separation technique that satisfies speed of analysis, selectivity, sensitivity, adequate throughput capacity, and affordable cost of analysis is unavailable yet. * To whom correspondence should be addressed. Tel: (404) 727-8027. Fax: (404) 727-3660. E-mail:
[email protected] or
[email protected]. † Departments of Surgery and Biomedical Engineering, Emory University School of Medicine and Georgia Institute of Technology. ‡ Department of Chemistry, Emory University. § School of Chemical and Biomolecular Engineering, Georgia Institute of Technology. 10.1021/pr050245g CCC: $30.25
2005 American Chemical Society
Bioaffinity chromatography has been widely used for the purification of biomolecules, in which affinity ligands, receptor or antibodies were immobilized onto solid supports such as sepharose beads, synthetic polymer resins, and silica gels.1 These approaches, however, have fundamental disadvantages resulting from the insolubilization of immobilized ligands. Particularly, the vibrational and rotational motions of biomolecules immobilized on solid surfaces are restricted, and in most cases, these restrictions reduce the affinity of the ligands for the target molecules as compared with free ligand. In addition, solid-liquid heterogeneous reactions are generally slower and less effective than homogeneous solution reactions due to restricted diffusion. Alternatively, affinity precipitation has been the focus of growing attention as an effective purification approach through selective recovery of target proteins by simple environmental triggers, such as temperature and pH.2 In principle, the target protein is selectively bound to a stimuliresponsive polymer-ligand conjugate in solution and removed from other extracts by precipitation. Purified proteins are recovered by disassociation from the polymer conjugate in solution again, which can be reused in repetitive cycles. In an initial demonstration of this approach, synthetic polymer, poly(N-isopropylacrylamide) (poly-NIPAM), which exhibits a thermally reversible phase transition, was developed by conjugating with DNA, antibody, proteins, such as streptavidin, or other smaller ligands.3 Despite the significance of poly-NIPAMs-based affinity precipitation systems, the use of poly-NIPAM often requires the implementation of complex bioconjugation schemes. Moreover, since most poly-NIPAM-based polymers display a relatively broad polydispersity, the precipitation efficiency of these conjugates may be somewhat compromised. In response to these limitations, monodisperse recombinant elastin-like polypeptides (ELPs) would be an alternative to polyNIPAM. ELPs consist of oligomeric repeats of the pentapeptide Val-Pro-Gly-Xaa-Gly and exhibit a reversible inverse temperature transition that is tunable through the choice of a “guest residue” Xaa.4 Recently, ELPs have been used as terminal tags in recombinant systems to facilitate recombinant protein purificaton,5 and have recently been conjugated to metalbinding ligands for affinity purification via temperature-triggered precipitation.6 Journal of Proteome Research 2005, 4, 2355-2359
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technical notes
Glyco-Affinity Precipitation Purification
Figure 1. Schematic illustration of one-pot glyco-affinity precipitation purification.
Cell-surface glycocojugates play important roles in cell-cell communication, proliferation, and differentiation through carbohydrate-protein interactions.7 Thus, studies of carbohydrate-protein interactions might provide new insights into their biological roles and reveal new possibilities for drug development.8 In the past decade, a diverse carbohydrate-based technology, termed glycotechnology such as glyco-arrays9 and glyco-affinity chromatography,10 have been developed as an advanced technology for functional studies of glycans. A guiding principle of these glycotechnologies is the display of carbohydrates onto a pertinent support or carrier that is often either a polymer,11 micro/nanoparticle,12 or solid surface.13 However, most of these constructs suffer the disadvantage of the insolubilization of immobilized ligands as well, and thus their efficiency or sensitivity were somewhat compromised. Herein, we report design and synthesis of an inverse thermalresponsive glyco-polypeptide polymer by recombinant and chemical techniques as a flexible carbohydrate array and demonstrate a one-pot glyco-affinity precipitation purification of carbohydrate-binding protein for enhanced proteomic. Notably, this strategy takes the advantages of both solutionphase capturing and releasing and solid-phase separation of target protein, which facilitate a simple and high efficient protein purification and further identification (Figure 1).
Experimental Section Reagent and Materials. All solvents and reagents were purchased from commercial sources and were used as received, unless otherwise noted. Synthesis of 2-aminoethyl β-glucoside (1) and 2-aminoethyl β-lactoside (2) were described in Supporting Information in detail. Neu5AcR2-3Galβ1-4(FucR13)GlcNAcβ1-3Galβ-O(CH2)3NH2 (3) was purchased from Lectinity Inc. (Moscow). Recombinant ELP triblock polypeptide (B9) comprised of flanking hydrophobic endblocks [(IPAVG)4(VPAVG)]16 and a hydrophilic midblock [(VPGVG)4(VPGEG)]48 was synthesized as our previous report.15 Synthesis of Glucose, Lactose and Sialyl Lewis x-Modified Elastin-Mimetic Triblock Polypeptide B9. To a solution of B9 (100 mg, 5.8 × 10-4 mmol) in 20 mM HEPES buffer (pH 6.0) was added 2 (53 mg. 0.139 mmol, 5 equiv.), hydroxysulfosuccinimide (Sulfo-NHS) (3 mg, 1.39 × 10-2 mmol), and 1-ethyl3-[3-dimethylaminopropyl]carbodiimide (EDC) (11 mg, 5.56 × 10-2 mmole) at 0 °C. The reaction mixture was stirred for 72 h at 4 °C and dialyzed against distilled water for 48 h at 4 °C to afford B9-Lact 5 (106 mg. 60%) after lyophilization. Similarly, modification of B9 (25 mg, 1.5 × 10-4 mmole) with 1 (8 mg, 0.035 mmol) and 3 (2 mg, 0.002 mmol) gave the glucose and 2356
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sialyl Lewis x-modified polypeptides B9-Glc 4 (26 mg, 58%) and B9-SLex 6 (2.3 mg, 66%), respectively. 1H NMR spectra of 4, 5, and 6 in D2O (10 mg/mL) were recorded using a Varian INOVA 600 spectrometer with magnetic field strengths of 600 MHz (detail in Supporting Information). Differential Scanning Calorimetry (DSC) of B9 and B9Lactose. Phase-transition temperatures and enthalpies were determined using a micro DSC III (Sterama, Inc.) differential scanning calorimeter. Heating and cooling aqueous samples at a rate of 10 °C/h between 10 and 60 °C with sample of B9 and B9-Lact in PBS buffer solution (10 mg/mL). Fluorescent Intensity Analysis. Fluorescent intensities of noncaptured and captured lectin-FITC in capturing and releasing PBS solution of B9 and B9-Lactose samples were determined using an ISS PCI Photocounting Spectrofluoremeter. For FITC, the excitation and emission wavelengths were set at 494 and 520 nm, respectively.
Results and Discussion We sought to design a carbohydrate ligand modified ELP sequence with a predicted Tt < 25°C (room temperature), so that all procedures for protein binding, precipitating, as well as releasing steps would be performed at low temperature to avoid target protein denaturing and detaching risk during the temperature-triggered precipitation process. It is well established that Tt of ELPs is strongly influenced by both the composition of the pentapeptide Val-Pro-Gly-Xaa-Gly and chain length.14 We have previously reported a high yield synthesis of a 170 kD recombinant ELP triblock polypeptide (B9) comprised of flanking hydrophobic endblocks [(IPAVG)4(VPAVG)]16 and a hydrophilic midblock [(VPGVG)4(VPGEG)]48.15 The endblocks were designed to undergo hydrophobic collapse at ∼22 °C, while the hydrophilic midblock exhibits a Tt that lies well above 37 °C, thereby segregating into compositionally, structurally, and spatially distinct domains. Thus, this copolymer affords micelles or larger network structures depending upon the solvent system, temperature, pH, and polymer concentration.15 Herein, we hypothesized that the thermally driven inverse transition temperature of the ELP-based triblock polymer would be maintained upon incorporation of carbohydrate ligands onto the hydrophilic midblock, and thus could be used as thermo-responsive glyco-affinity polymer for carbohydrate-binding protein purification. The presence of glutamic acid residues in the ELP midblock affords selective anchoring points for the incorporation of ligands or other probe molecules (Scheme 1). As model glycoligands, glucose, lactose, and sialyl Lewis x (SLeX) were chosen
technical notes
Sun et al.
Scheme 1. Synthesis of Glycosylated Elastin-Like Polypeptides
for carbohydrate-binding protein such as lectin16 and selectin17 detection and purification. As shown in Scheme 1, amine-linker derivatized glucose (1), lactose (2), and sialyl Lewis x (SLeX) pentasaccharide (3) were conjugated to ELP triblock polypeptide (B9) through formation of an amide linkage between glutamic acid and amine linker using carbodiimide chemistry to give glyco-polypeptide polymer 4, 5, and 6 in good yield, respectively. The successful sugar installations were confirmed by 1H NMR spectroscopy. In particular, conjugated sugar ratios were calculated by comparing the integration of protons of the sugar linker and polypeptide backbone of resultant glycopolypeptide polymers. As shown in Figure 2, comparison of methylene protons of the lactose ethyl linker with β-methyl protons of alanine in the endblocks of polypeptide of B9-Lact (5) indicated that 60% of glutamic acids were conjugated with lacotose. Similarly, 58% and 66% of glutamic acid residues were conjugated with glucose and sialyl Lewis x pentasaccharide in B9-Glc (4) and B9-sLex (6), respectively. B9-Lact (5) was chosen as a model glyco-ELP to evaluate the efficacy of these compounds in the affinity precipitation purification of carbohydrate binding proteins. As anticipated, both temperature-dependent differential scanning calorimetry (DSC) (Figure 3) and 1H NMR spectroscopy (Figure 4) of B9 and B9-Lact in PBS confirmed the presence of a sharp endothermic transition at 22 °C that was consistent with selective collapse of the peptide endblocks. Particularly, in the NMR spectroscopy, proton resonances of alanine and isoleucine that are associated with the hydrophobic endblocks are well resolved below 22 °C, but disappear above 22 °C, while those
Figure 2.
1H
NMR of B9-Lact (5) in D2O at 4 °C.
peaks characteristically associated with the hydrophilic central block and carbohydrate moieties are visible throughout this transition. These results proved that the thermally driven inverse transition temperature of the ELP-based triblock polymer is maintained upon incorporation of carbohydrate ligands and thus can be used for carbohydrate-binding protein purification. The glyco-ELP-based glyco-affinity precipitation purification was demonstrated by using B9-Lact 5 to target a β-galactose specific binding lectin. As shown in Figure 5, B9 and B9-Lact were incubated with a FITC-labeled galactose binding lectin (Arachis hypogae, 120 kDa, SIGMA) in PBS at 4 °C for 2 h, followed by 30 min incubation at 37 °C to induce glyco-ELP precipitation, respectively. The precipitates were separated from the supernatant by centrifugation at room temperature for 30 min. Effective lectin capture was only observed for B9Lact (Figure 5, 5Bb), while no perceptible lectin capturing observed for B9 (Figure 5, 5Ab). The captured lectin was released by dissolving the recovered pellet in cold PBS containing free lactose, incubated at 4 °C for 2 h, and followed by a second precipitation step. The specific lectin capturing and releasing efficiency of B9-Lact was investigated by fluorescent intensity analysis of the supernatant of lectin-FITC capturing and releasing step. Thereby, the capturing capacity was determined as 8.3 × 10-10 mole of FITC-lectin/mg of B9-Lact in treating with excess lectin (see later gel electrophoresis assay). Preliminarily, releasing capacity was 80% of the captured lectin in current 4 °C low temperature and 2 h incubation condition, which needs further optimization. On the other hand, trace of nonspecific capturing for B9 (1.2 × 10-11 mole of FITC-lectin/ mg) was detected in the supernatant of second precipitation, which might be due to the random encapsulation during the
Figure 3. Endothermic thermal transitions for B9 and B9-Lact revealed by DSC. Journal of Proteome Research • Vol. 4, No. 6, 2005 2357
technical notes
Glyco-Affinity Precipitation Purification
Figure 4. Temperature-dependent 1H NMR spectra identify the phase transition for B9 and B9-Lact.
Figure 6. 12% SDS-PAGE Gel. Capture specificity test with galactose specific (+: Arachis Hypogae, 120 kDa) and nonspecific (-: Concavalian A, 102 kDa) lectins. SC: Supernatant of Capturing, SR: Supernatant of Releasing. MM: Molecular Markers. * Excess lectin was used for all assays.
the capturing polymer. Indeed, the preliminary weight recovery assay showed there were 80% of B9 and 77% of B9-Lact recovered after two precipitation processes.
Conclusion
Figure 5. Thermally assisted glyco-affinity precipitation purification. ASb: supernatant of B9 with lectin in first precipitation; ASc: supernatant of B9 with lectin in second precipitation; BSb: supernatant of B9-Lact with lectin in first precipitation; BSc: supernatant of B9-Lact with lectin in second precipitation.
precipitation process. Nonetheless, the random captured protein in B9 was removable in an additional precipitation, which support it is nonspecific capturing. To assess capture specificity, both B9 and B9-Lact were incubated with a glactose-specific lectin (+: Arachis hypogae, 120 kDa) and a glucose/mannose specific lectin (-: Concavalin A, 102 kDa, SIGMA) and followed by thermal-precipitation, respectively. Subsequent incubation and precipitation in the presence of excess free lactose in combination with gel electrophoresis was performed to release and identify the specific captured lectin. As demonstrated in Figure 6, selective capture of the galactose-binding lectin was confirmed (Arachis hypogae: lane 8), while capture did not occur for Concavalin A (lane 10) with B9-Lact; nor was lectin capture observed for both Arachis hypogae (lane 4) and Concavalin ((lane 6) with the use of the nonderivatized B9. In addition, there were no observable ELP polypeptide (B9) and glyco-ELP polypeptide (B9-Lact) determined in either supernatant of capturing or releasing in this gel assay that demonstrates the precipitation efficiency of 2358
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In summary, we have demonstrated a one-pot glyco-affinity precipitation purification of carbohydrate-binding protein by designing thermal-responsive glyco-ELPs with selective affinity to carbohydrate-binding proteins. The biocapture potential is optimized by a flexible combination of multivalent carbohydrate-protein interactions and the thermal-responsive property of glyco-ELP polymer. Notably, both capture and release are conducted in solution, which improves the efficiency of these processes, whereas separation of the protein-captured glycoELP pellet and supernatant at low-temperature preserves target protein activity. By appropriate glyco-polypeptide polymer design, new opportunities might be provided for enhanced protein purification and identification, cell isolation, and pathogen detection applications.
Acknowledgment. This work was supported by Grants from the URC of Emory University and AHA for X.-L.S. and by grants from the NIH (E.L.C.). Supporting Information Available: Detail synthetic procedures for sugar monomers and spectral data for intermediates and final compounds (PDF). This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Chaiken, I. Bioaffinity chromatography - synergy between interactive chromatography and molecular recognition for the separation and analysis of macromolecules, J. Chromatogr. Biomed. Appl. 1989, 488, 145-160. (b) Sadana, S. D. A bioseparation using affinity techniques. J. Biotech. 1991, 19, 83-98. (c) Jungbauer, A. Chromatographic media for bioseparation. J. Chromatogr. A. 2005, 1065, 3-12. (2) Kim, J. Y.; Mulchandani A.; Chen, W. Temperature-triggered purification of antibodies. Biotechnol. Bioeng. 2005, 90, 373-379. (b) Carter, S.; Rimmer, S.; Sturdy, A.; Webb, M. Highly branched stimuli responsive poly[(N-isopropyl acrylamide)-co-(1,3-propandiol-3-methacrylate)]s with protein binding functionality. Macromol. Biosci. 2005, 5, 373-378. (c) Goerke, A. R.; TO, B. C. S.; Lee, A. L.; Sagar, S. L.; Konz, J. O. Development of a novel
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