Article Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Multifunctional, High Molecular Weight, Post-Translationally Modified Proteins through Oxidative Cysteine Coupling and Tyrosine Modification Brian M. Seifried, James Cao, and Bradley D. Olsen* Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States S Supporting Information *
ABSTRACT: Glycoproteins and their mimics are challenging to produce via chemical or biological methods because of their long protein backbones and large number of polysaccharide side chains that form a densely grafted protein−polysaccharide brush architecture. Herein, we demonstrate a new approach to protein bioconjugate synthesis that can approach the molar mass and functionalization densities of natural glycoproteins such as mucins and aggrecans. In this method, a tyrosine-enriched protein sequence is engineered and synthesized in E. coli, and sugars or other functional moieties can be efficiently and polyvalently grafted to the backbone through tyrosine modification chemistry. Cysteine residues on the chain ends are used for oxidative chain polymerization into high molar mass chains larger than can be easily expressed in the host. The effects of tyrosineenrichment and cysteine-incorporation on the physical and expression properties on a model protein are explored. Elastin-like peptides (ELPs) are chosen because of their high expression yields, repetitive sequence, substitutable amino acids, and wellstudied physical properties. The sequence modifications to mimic glycoproteins are shown to affect the maximum length of expressible sequence but not yield. The tyrosine modification chemistry is shown to functionalize up to 73% of all tyrosines on the peptide, and the scope of functional groups that can be mass conjugated to proteins is expanded through multistep conjugation strategies involving copper(I)-catalyzed alkyne−azide cycloaddition showing up to 97% alkyne functionalization. All of the functionalization chemistries preserve the ability to polymerize the backbone.
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INTRODUCTION Many proteins, such as mucins1 and aggrecans,2 are large glycoproteins that are functionalized at many sites along the protein backbone to form polymer brushes. Mucins act as adhesion decoys for viruses,3 sterically hinder pathogen transport across mucus layers,4 house antimicrobial molecules, and manage cell hydration.5 Aggrecans lubricate body joints,6,7 interact with hyaluronic acid,2 and help facilitate the ability of cartilage to resist compressive forces.8 The properties necessary to perform this rich assortment of functions require high molar mass and dense grafting with a diverse assortment of functional groups. Mucins are characterized as polydisperse, linear bottle brush polymers with molecular weights reaching several megadaltons.9 Mucins are densely functionalized with typically 25−30 carbohydrate chains per 100 amino acids10 that contain multiple functionalities such as N-acetylglucosamine, fructose, sialic acids, and sulfate.9 Similar to mucins, aggrecans are high molecular weight, bottlebrush-shaped and negatively charged. The core aggrecan protein is typically on the order of 300 kDa with glycosaminoglycan (GAG) side chains extending from the © XXXX American Chemical Society
glycosylated serine residues. The GAG side chains consist of polymer repeats of disaccharides composed of glucuronic acid and glycosamine with 40−50 disaccharide repeats per chain. Aggrecans have up to 100 GAG side chains per protein and an additional 30 chains per protein of keratan sulfate that are shorter chains consisting of 20−25 repeating units of galactose and N-acetyl glucosamine.7,11 The high molar masses and bottle brush structure of mucins and aggrecans lead to their characterization as stiff linear polymers with relatively long persistence lengths due to their heavy glycosylation and resulting dense negative charge.7 The bottle brush structure of these glycoproteins also significantly increases their entanglement molecular weights.12,13 In addition to entanglement, these glycoproteins behave like transient gels14,15 due to chemical cross-linking through cysteine bonds in mucins.7 Further, properties that are attributed to the side Received: December 30, 2017 Revised: April 22, 2018
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DOI: 10.1021/acs.bioconjchem.7b00834 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Figure 1. Illustration of synthetic strategy. Tyrosine enriched and cysteine-flanked ELP oligomers are functionalized via diazonium coupling. Alkyne functionalized ELPs from this method are further functionalized via CuAAC click chemistry.
reactive,38,39 and potentially receptive for multisite modification.40 These attractive properties suggest that tyrosine is an ideal candidate for protein mass-modification. In this work, we present a new platform for the synthesis of polyfunctional, high molar mass protein polymers that mimic the chemical structure of natural glycoproteins based on tyrosine bioconjguation and oxidative cysteine coupling. Protein polymers are engineered with cysteine end groups for oxidative chain extension, enabling expression and postpolymerization to produce very high molar mass protein backbones.31,41,42 The protein polymers have a high density of tyrosine residues, enabling site-specific conjugation that is orthogonal to the cysteine coupling reaction. The effects of tyrosine enrichment and cysteine flanking on proteins in terms of expression and physical properties are demonstrated on the well-studied elastin like peptide (ELP).43 A toolbox of highefficiency, high-specificity reactions focusing on diazonium coupling chemistry is demonstrated for the mass modification of tyrosine moieties that can also preserve cysteine oligomerization. Multistep conjugations utilizing copper(I) catalyzed alkyne−azide cycloaddition (CuAAC) as a second step are also demonstrated to expand functional diversity. This strategy is illustrated in Figure 1 and Scheme 1.
chains are enhanced by polyvalency; for example, backbone length has been shown to control quintessential glycoprotein properties such as virus adhesion.16 Other glycoprotein properties are a result of the dense functional group grafting. For example, highly negatively charged mucins will repel one another and change their behavior from that of a gel to a low viscosity lubricant.17 To create these high molecular weight, densely functionalized glycoproteins, cells typically synthesize a monomeric, lightly glycosylated precursor in the Golgi apparatus that is subsequently glycosylated by enzymes both inside and outside the cells.18 This process is complex and difficult to replicate synthetically. High molecular weight proteins typically express at lower yields and in slower expressing systems than smaller peptides.19 Further, the current understanding of peptide modifying enzymes is insufficient for mass modification purposes. Those challenges limit the potential of direct biological synthesis of mucins and most complex posttranslationally modified proteins.20 Researchers have hypothesized that the majority of glycoprotein properties can be mimicked by matching their functionality and size with synthetic bottlebrush polymer glycoprotein mimics. Some of the general strategies involve polymerizing a glycosylated synthetic monomer,16,21−24 polymerizing sugars25 with the option to affix small peptide units to them,26 or polymerizing a glycosylated amino acid.27−30 These strategies have been shown to be effective in mimicking properties of smaller glycoproteins such as cell surface mucins22 or the virus adhesion properties of natural mucins.16 The amino acid sequence of the backbone gives additional functionality, control over the placement of glycosidic groups, and the ability to oligomerize to reach very high molecular weights.31 The key challenge utilizing protein backbones is post-translationally functionalizing them.32 Researchers have developed a host of bioconjugation techniques that can modify cysteines,33 lysines,34 n-terminal amines,35 unnatural amino acids,36 and tyrosines.37 Among these targets, tyrosine moieties are specific,
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RESULTS AND DISCUSSION Design of Tyrosine-Enriched Protein Polymers. ELPs were chosen as a model protein to enrich with tyrosines and flank with cysteines because of their high expression levels in Escherichia coli (E. coli), known thermoresponsive behavior,43 repetitive sequence, and presence of a residue that could be easily substituted.43 A 3:1 ratio of tyrosine to serine was chosen for X in the VPGXG ELP pentapeptide repeat in an attempt to balance the hydrophobicity of the overall sequence.44 A 24 pentapeptide protein sequence was doubled three times between a His-tag and cysteine containing peptide sequence and c-terminal cysteine via restriction cloning and expressed in C41 cells to produce genes encoding for 24, 48, 96, and 192 B
DOI: 10.1021/acs.bioconjchem.7b00834 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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culture, respectively. These results suggest that tyrosine enrichment and cysteine flanking of ELP sequences does not have detrimental effects on expression levels compared to literature values.46 However, these sequence modifications reduce the maximum expressible length.47 These tyrosine enriched ELPs had a very high degree of oligomerization after oxidative cysteine-coupling compared to other protein sequences.41,42,48 Protein gels of partially reduced proteins demonstrating ladders of oligomerization are presented in Figure 2. Whereas in other sequences a protein ladder was clearly visible after oxidation for several days, the equilibrium distribution of oligomers was not resolvable by SDS-PAGE, and the fully oxidized, unmodified proteins would not dissolve in 6 M urea buffer at pH 5−11 until the addition of reducing agent. Samples that were completely cleaved via addition of reducing agent (200 equiv of Tris(2-carboxyethyl)phosphine (TCEP)) would reoligomerize within the time scale of sample preparation for electrophoresis if not sufficiently dilute (2 mg/mL). However, if reducing agent is not added to solution the proteins will precipitate out after several hours as
functionalities achievable via diazo coupling to functionalities such as sugars that are incompatible with the highly acidic environment required, a two-step diazonium coupling and CuAAC scheme was developed. The versatility of this strategy is demonstrated on PEO chains, galactose, and α-2,6-linked sialic acid. The limited water solubility of the alkyne functionalized proteins (>0.1 mg/mL) and the His-tag on the protein that will complex with copper ions53 create challenges in designing reaction conditions. Dilute protein (1 mg/mL) concentrations in DMF utilizing copper(I) halide with an excess of copper relative to the His-tag were found to be effective. FTIR was performed on the samples to ensure the diazonium bond was preserved during the click reaction (Figure 4). The extent of reaction was characterized by NMR because the samples would not fly by MALDI mass spectroscopy and E
DOI: 10.1021/acs.bioconjchem.7b00834 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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and sulfonate and PEO2000 functionalities were found to improve water solubility. Sensitive chemical groups such as saccharides like sialic acid and galactose that are incompatible with the acidic conditions present in diazonium coupling were mass conjugated to the protein through a two-step utilizing diazonium coupling followed by copper(I)-catalyzed alkyne− azide cycloaddition, which had nearly quantitative yield. Oxidative coupling were preserved even after functionalization for all reactions studied as measured by SDS-PAGE. This combination of techniques allowed for the creation of multiply functionalized high molecular weight protein brushes that mimic natural glycoproteins.
they return to their fully oxidized state. Heavily oligomerized peptides can be dissolved in solution with the addition of reducing agent and agitation. Chain Oligomerization of Functionalized Proteins. All of the different bioconjugations preserved the ability of the proteins to undergo oxidative chain coupling, forming high molar mass proteins. Conjugating water-soluble side chains at multiple sites allows the conjugate products to be dissolved in water. This allowed the sulfonate, galactose, 6SA, and PEO functionalized product to be tested for compatibility with cysteine oligomerization through SDS-PAGE. However, the equilibrium distributions of the oligomerized products were either no longer water-soluble or not resolvable via SDS-PAGE. The effectiveness of the cysteine oligomerization in creating very high molecular weights is presented in Figure 5 through showing oxidized and partially reduced versions of the products. Fully reduced versions are presented in Figure S25. The sulfonate functionalized protein was imaged without staining because of the protein’s bright orange color. Both PEO functionalized proteins exhibited very high oligomerized molecular weights with some bands not even moving on the protein gel. However, it is known that PEO chains have inconsistent effects on readings on SDS-PAGE and exhibit significant smearing.54 The sugar functionalized peptides were imaged with a more sensitive silver stain, because Coomassie blue was not sensitive enough to image the sialic acid functionalized moiety. The alkyne functionalized protein was not water-soluble, even in the fully reduced state, and could not be characterized via SDS-PAGE. Regardless, functionalized proteins with molecular weights greater than 250 kDa are observed. The solvent fronts were not fully run off the gel to make sure there was no unfunctionalized peptide being run off the gel. The solvent fronts do not contain peptides as shown in Figure S26 where the front is visible by the silver stain but not by Coomassie blue. The purity of the conjugates were checked via SEC. E24, E24−6SA, and E24-Gal showed no small molecule impurities both before and in the solvent regions. E24-Sulfonate, E24-PEO2000Ether, and E24-PEO2000Click all interacted with the column. These results are presented in Figure S27.
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METHODS Instrumentation and Characterization. Matrix-assisted laser desorption/ionization time-of- flight (MALDI-TOF) mass spectra were obtained on a Bruker Microflex instrument equipped with a 337 nm nitrogen laser in the Koch Institute at MIT. α-Cyano-4-hydroxycinnamic acid (CHCA) was used as matrix. Protein NMR spectra were recorded on an INOVA 500 MHz spectrometer. The residual undeuterated solvent peaks were used for reference. The following abbreviations are used to denote the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. Coupling constants J are reported in Hertz (Hz). Visualization of protein bands was accomplished by staining with Coomassie Brilliant Blue R-250 (Fisher). Silver stains were performed using the PROTEOSILVER stain kit from SigmaAldrich. Commercially available markers (Color Prestained Protein Standard, New England Biolabs) were applied to at least one lane of each gel. Immunostains were performed with the Mini Trans-Blot module for the Mini-PROTEAN Tetra cell system following literature protocols. Sample was resolved by 12 wt % SDSPAGE. Proteins were transferred to nitrocellulose membrane and then blocked with 5 wt % nonfat dry milk for 1 h. Histagged proteins were bound with G020 antibody (1:1000 dilution) and a secondary Anti-Mouse IgG-alkaline phosphatase from Sigma-Aldrich (1:1000 dilution). A color-development solution (mix BCIP solution 200 μL, (BCIP 150 mg, DMF 10 mL to make a 10 mL stock), NBT solution 200 μL, (NBT 300 mg, water 3 mL, DMF 7 mL to make a 10 mL stock), Development buffer 20 mL (Tris 6.05 g, MgCl2·6H2O 0.06 g, water 500 mL, to make a 500 mL stock) was added to detect the proteins and the reaction was quenched with distilled water.55 UV−vis spectroscopy was performed with the Implen NanoPhotometer P330 using a quartz nanodrop cell with a 1 mm path length. Fourier transform infrared spectroscopy was taken on a Thermo Fisher FTIR6700 of potassium bromide pellets of the samples in transmission mode with 32 sample scans and 32 background scans. Pellets consisted of 0.5 wt % protein pressed in potassium bromide in a carbon press. Size-exclusion chromatography (SEC) was performed on an Agilent 1260 system with an Agilent SEC-3 column. The mobile phase was 100 mM PBS pH 7.4 in Milli-Q water. The LS detector used was a Wyatt Dawn 8+. Gene Construction and Protein Expression. Genes for the tyrosine enriched ELP sequences were cloned in a stepwise fashion that allowed for the efficient doubling of the molecular weight of the sequence. (Figure 6, Supporting Information). In brief, ELP genes were synthesized with modular restriction sites that allowed for sequential digestion and insertion of ELP
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CONCLUSIONS Many of the properties of glycoproteins like aggrecans and mucins stem from their high molecular weight and dense functionalization. These two features are challenging to reproduce with protein systems. Through this work, we presented a scalable and efficient approach to creating high molecular weight heavily modified protein brushes via the combination of cysteine and diazo coupling reactions. The tyrosine enrichment required to prepare ELP proteins for multifunctional modification prevented protein expression at 96 pentapeptide repeats and above, but maintained yields comparable to other work46 at 48 repeats and below. The LCST behavior of the protein was moved out of the experimental range and they were insoluble at 4 °C for all molar masses studied. Tyrosine was demonstrated as an attractive target for mass bioconjugation for protein because of the high specificity and efficiency of the diazonium coupling reaction. The technique was performed in reducing and denaturing conditions as well as shown to couple chemical functionalities in sufficient quantities to affect the protein solubility as well as orthogonal to cysteine coupling. Alkyne functionality was found to improve organic solvent solubility, F
DOI: 10.1021/acs.bioconjchem.7b00834 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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dialyzed against ultrapure water to remove small-molecule reagents, and then lyophilized. Measurements of Conversion. For UV−vis measurement, samples were massed and dissolved in solution at 1 mg/ mL. E24, E24 Sulfonate, and E24 PEO2000 Ether were dissolved in 6 M Urea 150 mM PBS, pH 9, and 1% vol βME aqueous buffer. E24 Alkyne, E24 Galactose, E24 6SA, and E24 PEO2000 Click were dissolved in DMF with 150 mM acetic acid and 1% vol βME. The UV−vis spectra of the samples were taken and the mass of functional group were calculated using the molar extinction coefficients measured from the small molecule analogues. Boc-tyr-sulfonate was used as a model for E24-sulfonate, boc-tyr-alkyne was used for E24-alkyne, boc-tyralcohol was used for E24-PEO2000Ether, and boc-tyr-OPEO was used as a model for E24-galactose, E24−6SA, and E24PEO2000Click. The mass of the functional group was subtracted from the total mass to yield the mass of proteins. The mols of functional group was divided by the mols of protein to get the average number of functional groups per protein. This total was divided by the number of tyrosines per protein (18 for E24).
Figure 6. Graphical representation of cloning strategy.
sequences using restriction enzymes. The final sequence was directly ligated into a PET15b vector for expression. Gene and protein sequences are included in the Supporting Information. The proteins were expressed in Escherichia coli (E. coli) strain C41. The cells were grown in 1 L TB cultures in Fernbach flasks at 37 °C until reaching OD600 = 1.0. Expression was induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside. The cells were harvested 18 h after induction and resuspended in 100 mL lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10 mM βME, adjusted to pH 8.0 with NaOH). The cells were lysed through two freeze−thaw cycles at −80 °C followed by sonication for 3 × 10 min. The solution was centrifuged and the solids were collected. The solids were then incubated in 100 mL denaturing buffer (8 M Urea, 20 mM Tris, 10 mM βME, adjusted to pH 8.0 with NaOH) at 37 °C with shaking for 4 h. The solution was centrifuged, and the liquid fraction was collected. The proteins were first purified by precipitation in 5 wt % ammonium sulfate. The solids were collected and redissolved in 100 mL denaturing buffer. The ELP was subsequently purified via metal affinity chromatography using Ni-NTA resin (Qiagen). The solids were collected via centrifugation and purity was confirmed by SDSPAGE and MALDI-TOF. Typical yields after purification were around 380 mg L−1 for E24 and 350 mg L−1 for E48. General Diazonium Coupling Protocol. Protocols for diazonium coupling were adapted from the literature.38 Protein (5 mg/mL) was dissolved in 6 M Urea, 500 mM Trizma Base, 10 mM βME, pH 9, by shaking for 12 h at 4 °C. The diazonium salt was prepared by dissolving the aniline precursor (4aminobenzenesulfonic acid, 4-ethynylaniline, or PEO2000aniline) at 20 mg/mL into p-TsOH (160 mg/mL). The solution was mixed with NaNO2 (32 mg/mL, 1.6 eq relative to aniline concentration), vortexed for 1 min, and allowed to react at 4 °C for 1 h. Then 15 eq. relative to the tyrosine concentration of the azo salt was added to the protein solution, vortexed for 1 min, and stirred at 4 °C for 12 h. The reaction mixture was dialyzed against ultrapure water to remove small-molecule reagents and lyophilized. E24-Alkyne was dialyzed against ultrapure water, then acetone, then ethanol, and then ultrapure water again to remove water-insoluble small-molecule reagents before lyophilization. The higher number of equivalents and use of dialysis results in a better conversion than the small molecule analogues. General Copper(I) Assisted Alkyne−Azide Cycloaddition. Alkyne functionalized protein (10 mg, 0.59 μmol), azide species (1.1 equiv relative to the alkyne functional groups, 8 μmol), and PMDETA (0.75 equiv, 1.11 μL) were dissolved in anhydrous DMF (10 mL) in a vial. The solution was degassed by sparging with nitrogen for 30 min. Copper(I) bromide (0.75 eq, 0.76 mg) was added to start the reaction, and the solution was stirred 12 h under nitrogen. The reaction mixture was dialyzed against 10 mM Trizma, 50 mM NaCl, 0.01 mM EDTA pH 8 in ultrapure water to remove the copper catalyst, next
M total = Mbackbone + M functional group M functional group = wfunctional groupcV A = εbc
Mtotal = Sample mass. Mbacbkbone = Protein backbone mass. Mfunctional group =mass of functional groups. wfunctional group = Molar mass of functional group. c = Concentration. V = Volume. A = Absorbance. ε = Molar extinction coefficient. b = Path length. For measurement by MALDI (Figures S10 and S11), the mass of the protein was subtracted from the average molar mass by MALDI. This difference was divided by the molecular weight of the functional group added to result in the average number of functional groups per protein. This total was divided by the number of tyrosines per protein (18 for E24). The conversion of the diazonium coupling functionalized proteins as measured by NMR was calculated by comparing the integrations of unfunctionalized tyrosine protons (6.7 and 7.1 ppm) to functionalized tyrosine peaks. The chemical shifts of the unfunctionalized tyrosine peaks were identified through proton NMR of unfunctionalized E24 in d-DMF. This technique proves to be a robust method of determine conversion because tyrosine is the only aromatic amino acid in the backbone and the protons added through diazonium coupling have higher chemical shifts. The CuAAC conversions were calculated through first adding the total integrations of tyrosine peaks (6.6 to 7.6 ppm). That sum was compared to the integration of the hydrogens adjacent to the triazole (around 5.7 and 5.1 ppm) using the 50% alkyne functionalization as determined through NMR analysis of the precursor molecule.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00834. Materials, instrumentation and characterization, cloning strategy, peptide sequences, synthetic procedures, and additional spectra (PDF) G
DOI: 10.1021/acs.bioconjchem.7b00834 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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(9) Roussel, P., Lamblin, G., Lhermitte, M., Houdret, N., Lafitte, J.-J., Perini, J.-M., Klein, A., and Scharfman, A. (1988) The complexity of mucins. Biochimie 70, 1471−1482. (10) Lai, S. K., Wang, Y. Y., Wirtz, D., and Hanes, J. (2009) Microand macrorheology of mucus. Adv. Drug Delivery Rev. 61, 86−100. (11) Bansil, R., and Turner, B. S. (2006) Mucin structure, aggregation, physiological functions and biomedical applications. Curr. Opin. Colloid Interface Sci. 11, 164−170. (12) Verduzco, R., Li, X., Pesek, S. L., and Stein, G. E. (2015) Structure, function, self-assembly, and applications of bottlebrush copolymers. Chem. Soc. Rev. 44, 2405−2420. (13) Hu, M., Xia, Y., McKenna, G. B., Kornfield, J. A., and Grubbs, R. H. (2011) Linear Rheological Response of a Series of Densely Branched Brush Polymers. Macromolecules 44, 6935−6943. (14) Bansil, R., Stanley, E., and LaMont, J. T. (1995) Mucin biophysics. Annu. Rev. Physiol. 57, 635−57. (15) Celli, J., Gregor, B., Turner, B., Afdhal, N. H., Bansil, R., and Erramilli, S. (2005) Viscoelastic properties and dynamics of porcine gastric mucin. Biomacromolecules 6, 1329−33. (16) Tang, S., Puryear, W. B., Seifried, B. M., Dong, X., Runstadler, J. A., Ribbeck, K., and Olsen, B. D. (2016) Antiviral Agents from Multivalent Presentation of Sialyl Oligosaccharides on Brush Polymers. ACS Macro Lett. 5, 413−418. (17) Cone, R. A. (2009) Barrier properties of mucus. Adv. Drug Delivery Rev. 61, 75−85. (18) McCool, D. J., Forstner, J. F., and Forstner, G. G. (1994) Synthesis and Secretion of Mucin by the Human Colonic Tumor-Cell Line LS180. Biochem. J. 302, 111−118. (19) Graslund, S. (2008) Protein production and purification. Nat. Methods 5, 135−146. (20) Becker, T., Dziadek, S., Wittrock, S., and Kunz, H. (2006) Synthetic glycopeptides from the mucin family as potential tools in cancer immunotherapy. Curr. Cancer Drug Targets 6, 491−517. (21) Parthasarathy, R., Rabuka, D., Bertozzi, C. R., and Groves, J. T. (2007) Molecular orientation of membrane-anchored mucin glycoprotein mimics. J. Phys. Chem. B 111, 12133−5. (22) Chen, X., Lee, G. S., Zettl, A., and Bertozzi, C. R. (2004) Biomimetic engineering of carbon nanotubes by using cell surface mucin mimics. Angew. Chem., Int. Ed. 43, 6111−6116. (23) Kramer, J. R., and Deming, T. J. (2010) Glycopolypeptides via Living Polymerization of Glycosylated-l-lysine N-Carboxyanhydrides. J. Am. Chem. Soc. 132, 15068−15071. (24) Hudson, K. L., Bartlett, G. J., Diehl, R. C., Agirre, J., Gallagher, T., Kiessling, L. L., and Woolfson, D. N. (2015) Carbohydrate− Aromatic Interactions in Proteins. J. Am. Chem. Soc. 137, 15152− 15160. (25) Lee, S.-G., Brown, J. M., Rogers, C. J., Matson, J. B., Krishnamurthy, C., Rawat, M., and Hsieh-Wilson, L. C. (2010) Endfunctionalized glycopolymers as mimetics of chondroitin sulfate proteoglycans. Chem. Sci. 1, 322−325. (26) Bernhard, J. C., and Panitch, A. (2012) Synthesis and characterization of an aggrecan mimic. Acta Biomater. 8, 1543−1550. (27) Tachibana, Y., Matsubara, N., Nakajima, F., Tsuda, T., Tsuda, S., Monde, K., and Nishimura, S. I. (2002) Efficient and versatile synthesis of mucin-like glycoprotein mimics. Tetrahedron 58, 10213− 10224. (28) Deming, T. J. (2016) Synthesis of Side-Chain Modified Polypeptides. Chem. Rev. 116, 786−808. (29) Engler, A. C., Bonner, D. K., Buss, H. G., Cheung, E. Y., and Hammond, P. T. (2011) The synthetic tuning of clickable pH responsive cationic polypeptides and block copolypeptides. Soft Matter 7, 5627−5637. (30) Oelker, A. M., Morey, S. M., Griffith, L. G., and Hammond, P. T. (2012) Helix versus coil polypeptide macromers: gel networks with decoupled stiffness and permeability. Soft Matter 8, 10887−10895. (31) Tang, S., Glassman, M. J., Li, S., Socrate, S., and Olsen, B. D. (2014) Oxidatively Responsive Chain Extension to Entangle Engineered Protein Hydrogels. Macromolecules 47, 791−799.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Bradley D. Olsen: 0000-0002-7272-7140 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Defense Threat Reduction Agency under contract HDTRA1-13-1-0038 and made use of the MRSEC Shared Experimental Facilities at MIT, supported by the National Science Foundation under award number DMR-1419807. The authors would also like to thank Dr. Minkyu Kim, Dr. Yun Jung Yang, Dr. XueHui Dong, Dr. Allie Obermeyer, and Dr. Bruno Silveira De Souza for helpful conversations and Wui Yarn Chan for assistance with the FTIR measurements. The authors report no conflicts of interest.
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ABBREVIATIONS ELPs, elastin like peptides; GAG, glycosaminoglycan; E. coli, Escherichia coli; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TCEP, Tris(2-carboxyethyl)phosphine; Boc-tyrosine, N-(tert-butoxycarbonyl)-L-tyrosine; OPEO, oligopoly(ethylene oxide); HPLC, high-performance liquid chromatography; NMR, nuclear magnetic resonance; FTIR, Fourier-transform infrared; PEO, poly(ethylene oxide); DMF, dimethylformamide; CuAAC, copper(I)-catalyzed alkyne−azide cycloaddition; MALDI-TOF, matrix assisted laser desorption ionization-time-of-flight; UV−vis, ultraviolet−visible; 6SA, α-2,6-linked sialic acid; LCST, lower critical solution temperature; CHCA, α-cyano-4-hydroxycinnamic acid; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; NBT, nitro blue tetrazolium chloride; NaOH, sodium hydroxide; βME, 2mercaptoethanol; p-TsOH, p-toluenesulfonic acid; NaNO2, sodium nitrite; PMDETA, N,N,N′,N″,N″-pentamethyldiethylenetriamine; NaCl, sodium chloride; EDTA, ethylenediaminetetraacetic acid; PBS, phosphate-buffered saline; ppm, parts-per million
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REFERENCES
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DOI: 10.1021/acs.bioconjchem.7b00834 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.bioconjchem.7b00834 Bioconjugate Chem. XXXX, XXX, XXX−XXX
