Antibody-binding, anti-fouling surface coatings based on recombinant

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Antibody-binding, anti-fouling surface coatings based on recombinant expression of zwitterionic EK peptides Julia Ann-Therease Walker, Kye J. Robinson, Christopher Munro, Thomas Gengenbach, David A. Muller, Paul R. Young, Linda HL Lua, and Simon Robert Corrie Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00810 • Publication Date (Web): 25 May 2018 Downloaded from http://pubs.acs.org on May 26, 2018

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EK PEPTIDE FILMS FOR CONTROLLED BIO/NANO INTERACTIONS

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Antibody-binding, anti-fouling surface coatings based on recombinant expression of zwitterionic EK peptides

Julia A. Walker, ╪ Kye J. Robinson, ╪ Christopher Munro, † Thomas Gengenbach, ℓ David A. Muller, ∩ Paul R. Young, ∩ Linda H. L. Lua† and Simon R. Corrie *╪

╪ Department of Chemical Engineering, Monash University, Clayton, Victoria, 3800, ARC Centre of Excellence in Convergent BioNano Science and Technology, Monash Node. † The University of Queensland, Protein Expression Facility, Brisbane, QLD 4072, Australia. ℓ CSIRO Manufacturing, Clayton, VIC 3168, Australia ∩ The University of Queensland, School of Chemistry and Molecular Biosciences, Brisbane, QLD 4072, Australia

The authors acknowledge funding from the Australian Research Council (CE140100036). *[email protected]


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Abstract Development of anti-fouling films which selectively capture or target proteins of interest is essential for controlling interactions at the “bio/nano” interface. However, in order to synthesise bio-functional films from synthetic polymers that incorporate chemical “motifs” for surface immobilization, anti-fouling, and oriented biomolecule attachment, multiple reaction steps need to be carried out at the solid/liquid interface. EKx is a zwitterionic peptide that has previously been shown to have excellent anti-fouling properties. In this study, we recombinantly expressed EKx peptides and genetically encoded both surface attachment and antibody-binding motifs, before characterised the resultant biopolymers by traditional methods. These peptides where then immobilised to organosilica nanoparticles for binding IgG, and subsequently capturing dengue NS1 as a model antigen from serum-containing solution. We found that a mixed layer of a short peptide (4.9 kDa) backfilled with a longer peptide terminated with an IgG-binding Z-domain (18 kDa) demonstrated selective capture of dengue NS1 protein down to ~10 ng ml-1 in either PBS or 20% serum.

Keywords: Zwitterionic peptides, silica nanoparticles, antifouling, selective capture, Dengue NS1.


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[Title Here, up to 12 Words, on One to Two Lines] Controlling the interactions between synthetic materials and biological components requires both high target selectivity and minimal non-specific binding. Target selectivity is commonly achieved through the immobilization of antibodies, peptides, and other biomacromolecules to the material, while reduction of non-specific binding is achieved by coating surfaces with hydrophilic polymer films. The most commonly employed polymer to date is poly(ethylene glycol) (PEG), based on its broad coverage in the literature and the commercial availability of a range of PEG-containing polymers and reagents.1 Zwitterionic polymers and peptides have recently emerged, which have been shown to form a stronger hydration layer compared to PEG in some contexts, and which may have advantages for in vivo applications in terms of biodegradability and reduced immunogenicity.2 Regardless of the polymer composition employed, targeting molecules need to be chemically immobilized. Moreover, many congugation chemistries negatively affecting the stability and/or function of the probes.3, 4 Thin films combining “motifs” for surface attachment, anti- fouling and targeting agent binding, in a sequence-controlled manner, are therefore highly desirable. While free radical and solid-phase synthesis methods are favoured for production of high quality films for biomedical coatings,5 recombinant approaches offer the advantage of sequence-control at the monomer level. “PASylation” is a recent example, in which amino acid sequences (random combination of proline, alanine and serine) are added to the N- or Cterminus of a protein in order to modify its biodistribution properties in vivo. Skerra et al showed that PASylation of interferon or Fab fragments resulted in significantly enhanced biological half-life and reduced organ clearance, similar to that of PEG-conjugates, however the PASylated proteins were produced from microbial cultures without the need for additional chemical modification.6 In another example, Jiang et al introduced zwitterionic ‘EK’ peptides as antifouling surface coatings in 2009, composed of alternating units of lysine and glutamic


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acid.7 The authors demonstrated that EK coatings impart low fouling properties in a biological environment on a gold surface. Nowinski et al showed that the addition of different terminal groups onto the EK peptide, could be used for surface attachment (proline tag) and cell binding (RGD peptide), while maintaining the anti-fouling characteristics of the EK segment.8 In 2015, Jiang et al also incorporated EK tags into two common enzymes and demonstrated retention of unmodified enzyme activity over long time periods at elevated temperature and ionic strength.9 In other work, Cui et al showed alternative methods of covalently attaching EK onto surfaces other than gold, using polydopamine mussel inspired chemistry.10 Recently, Wang et al in 2017 produced an electrochemical sensor, using the EK motif for antifouling and attaching a DNA probe via EDC-mediated amide crosslinking.11 However, to date, the recombinant production of polymers to form thin film coatings incorporating surface attachment, anti-fouling, and functional antibody-binding motifs has not been demonstrated. Protein engineering through recombinant approaches allows the synthesis of a DNA, protein or peptide sequence with monomer-level precision. In this study, we designed several simple peptides that contain surface-reactive, anti- fouling and bioconjugation motifs in the one sequence. Herein, we will describe the results of a screening process to express and purify peptides in high yield, along with results on surface attachment, anti-fouling, oriented antibody binding, and specific viral antigen (dengue virus NS1 protein) capture as a model system, using previously developed polymer-coated surfaces (SilanePEG-COOH) as controls. Materials and methods EK peptide plasmid construction EK1, EK2 and EKZ (collectively referred to as “EKx”) sequences were codon optimised for expression in Escherichia coli and synthesised by GeneArtTM (ThermoFisher Scientific). EK1 and EK2 were cloned into a modified pET-48b(+) vector containing a 6xHis


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affinity tag, maltose binding protein (MBP) solubility tag and tobacco etch virus protease (TEVP) recognition site upstream of the gene insertion site. EK1 and EK2 were ligated into SacII and EcoRI linearised vectors using T4 DNA ligase to generate 6xHis-MBP-TEVP-EK1 and 6xHis-MBP-TEVP-EK2. These MBP tagged constructs were linearised using NdeI and SacII to subclone 6xHis-Thioredoxin (TRX) or 6xHis fusion tags via homologous recombination. Z-domain was ligated into 6xHis-TEVP-EK2 linearised with SacII and EcoRI using T4 DNA ligase to generate 6xHis-TEVP-Z-EK2. All constructs were sequence verified by the Australian Genome Research Facility. EK peptide expression optimization Constructs were transformed into BL21(DE3)pLysS or BL21 StarTM(DE3) competent E. coli cells (ThermoFisher Scientific). Cultures of 0.5 mL Overnight ExpressTM Instant TB Medium (Merck) (OnX) containing 15 µg/mL kanamycin were inoculated and grown at 15˚C or 25˚C overnight (O/N). Cultures of 0.5 mL TB medium containing 15 µg/mL kanamycin were inoculated and grown at 25˚C, 400 rpm until OD600 = 0.5, whereat they were induced with 0.2 mM IPTG and incubated for 3 h (at 25˚C or 15˚C) or overnight (15˚C only). Cell pellets obtained from each culture condition were lysed in BugBuster® Protein Extraction Reagent (Merck) and centrifuged at 3,200 g for 45 min. The soluble fraction was incubated with loose Ni Sepharose Fast Flow resin (GE Healthcare). Unbound protein was removed with wash buffer (20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 8.0) and bound protein was eluted with elution buffer (20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 8.0). Scale-up EK peptide expression and purification Constructs were transformed into BL21 StarTM(DE3) competent E. coli cells. Cultures of 500 mL Overnight ExpressTM Instant TB Medium containing 15 µg/mL kanamycin in 2.5 L TunairTM shake flasks (IBI Scientific) were inoculated at a ratio of 1:50


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from an overnight starter culture and grown at 25˚C for 24 h before harvesting by centrifugation at 9,000 g for 20 min at 4˚C. All subsequent steps were performed at 4˚C, except where noted. Cell pellets from 500 mL expression cultures were resuspended in 10 mL lysis buffer per gram wet cell weight (20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, 1 % Triton X-100, cOmpleteTM Protease Inhibitor Cocktail (Roche Diagnostics), pH 8.0), lysed using a Branson Sonifier on ice and centrifuged at 14,000 g for 30 min. Soluble fractions were loaded onto 5 mL HisTrap FF Crude columns (GE Healthcare) preequilibrated with wash buffer. Unbound protein was removed with 10 column volumes (CV) wash buffer and bound protein was eluted in 5 CV elution buffer. Eluted protein was buffer exchanged into wash buffer using HiPrep 26/10 Desalting Columns (GE Healthcare) and digested with TEV protease using optimised tag removal conditions (1:10 (w/w) protease to substrate ratio overnight at 4˚C (EK1) or room temperature (EK2, EKZ)). Immediately following tag removal, samples were loaded onto a 5 mL HisTrap FF Crude column and purified peptides were collected in reverse mode in the flow through and wash fractions. Purified peptides were buffer exchanged into PBS, passed through a 0.2 µm filter and stored at -80˚C. SDS-PAGE analysis did not indicate any significant peptide loss after one freezethaw cycle. EK peptide analysis Peptides samples were analysed on NuPAGE 4-12 % Bis-Tris SDS-PAGE Gels (ThermoFisher Scientific) under denaturing and reducing conditions as per the manufacturer’s instructions, except where noted. Gels were visualised by SimplyBlue SafeStain (ThermoFisher Scientific) and imaged using a ChemiDoc XRS+ System (Bio-Rad). Target protein concentration was initially estimated by absorbance at 280 nm using a NanoDrop 1000 Spectrophotometer (ThermoFisher Scientific) (EKZ) or band intensity analysis by comparison to a standard of known concentration (EK1, EK2), and then


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confirmed by quantifying the free thiol concentration pure peptide solution using Ellman’s Reagent (Sigma Aldrich) after TCEP reduction of residual disulphide adducts. EK peptide immobilization to particles Prior to use EK1, EK2 and EKZ were incubated with Tris(2-carboxyethyl)phosphine hydrochloride (TCEP; Sigma Aldrich) in PBS using 10 molar equivalents for 10 min at room temperature. The EKx where then placed into individual 3000 kDa Amicon® Ultra spin columns (Sigma Aldrich), spun at 7500 RCF at 21 C ̊ washed, until no TCEP was detected in the wash by Ellman’s test (Sigma Aldrich). 400 nm, thiol-functionalised and Cy5-labelled nanoparticles (“MPS-Cy5 nanoparticles”) were synthesised according to the method of Vogel et al 2007.12 or immobilization reactions, 4 ml of 1.27x107 particles/mL MPS-Cy5 nanoparticles were incubated with 1.47 x 10-7 M EKx peptides in a 2:1 ethanol/water mixture, followed by 10 min of sonication and 24 hr incubation on an orbital shaker at room temperature, and thorough rinsing in Milli Q water for storage at 4 ̊C (resultant samples referred to as “MPS-Cy5@EKx nanoparticles”). To create mixed topology of EK1+Z and EK2+Z, nanoparticles with EK1 and EK2 surface coating where then incubated with EKZ (6 µL, 2.27X10-5 M) again for 24 hrs on an orbital shaker with all samples being washed three times with Milli-Q to remove the excess EKx and then suspended into PBS. PEG control coating For MPS@PEG control samples, 35 mg Silane-PEG-COOH (1kDa, Nanocs) was dissolved in 1 ml of 24 % w/w ammonia for base-catalysed crosslinking of silanol groups; these samples were incubated at room temperature for 24 h and rinsed thoroughly in Milli Q water before storage at 4 C ̊ . Antibody and NS1 capture onto EK-modified particles Anti-NS1-IgG (unmodified or labelled with A488) was diluted to 3.5 µg mL-1 in PBS buffer (0.01M) and incubated with 7.62 x 105 of MPS-Cy5@EKx particles then incubated at


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37 ̊C for 2 h following washing three times with PBST. For NS1 capture, NS1 was incubated with MPS-Cy5@EKx particles in PBS or PBS containing 10 % FBS (with or without bound anti-NS1-IgG), vortexed for 30 sec, and incubated at 37C on a shaker for 2 h. The samples were then centrifuged at 4000 RCF for 3 min and washed with PBST three times, and finally resuspended in PBS. The titration experiment was conducted using serial NS1 dilutions (ranging from 100-1.2 ng mL-1, based on previous studies)13,14,15 using either PBS or PBS/10 % FBS as the sample matrix. Non-specific NS1 protein absorption. NS1 protein was labelled with Alexa Flour 488 (A488, Thermo Fisher Scientific) according to the manufacturer’s protocol. The labelled protein was diluted to 100 ng ml1

(well above saturation for comparative bioassays)15 in PBS buffer then incubated with 7.62 x

105 MPS-Cy5@EKx particles at 37 ̊C for 2 h following washing three times with PBST (PBS containing 0.02 % tween) for flow cytometry analysis. 1

H NMR

Protein samples was taken on a Bruker Avance III 400 MHz NMR spectrometer in deuterated water (D2O). The spectra was analysed using Bruker TopSpin™ with the molecular weight determined by integrating the terminal protons by chain protons. Proton NMR assignments for EK1: 1H NMR (D2O, 400MHz) δ: 1.54(39H, b), 1.76(38H, b), 1.92(38H, b), 2.13(38H, b), 2.37(38H, b), 3.05(39H, b), 3.75(2H, b), 3.93(2H, b), 4.08(2H, b), 4.28(38H, b). Proton NMR assignments for EK2:1H NMR (D2O, 400MHz) δ:1.54(92H, b), 1.64(93H, b), 1.99(92H, b), 2.21(89H, b), 2.43(94H, b), 3.03(92H, t), 3.75(2H, b), 3.97(2H, b), 4.09(2H, b), 4.21(92H, b) Mass Spectrometry Protein samples were analysed by LC-MS using a quadrupole TOF mass spectrometer (MicroTOFq, Bruker Daltonics, Bremen, Germany) coupled online with a 1200 series


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capillary HPLC (Agilent technologies, Santa Clara, CA, USA). Samples injected onto a SGE C4 1000A (SGE, Melbourne, Australia) reversed phase column with 95 % buffer A (0.1 % Formic acid) at a flow rate of 7 µL min-1. The peptides are eluted over a 30-min gradient to 70 % B (80 % Acetonitrile 0.1 % formic acid). The eluant is nebulised and ionised using the Bruker electrospray source using the Low flow electrospray needle with a capillary voltage of 4500 V dry gas at 180 ºC, flow rate of 4 µL min-1 and nebuliser gas pressure at 300 mbar. Prior to analysis, the qTOF mass spectrometer was calibrated using 1:50 dilution tuning mix (Agilent technologies, Santa Clara, CA, USA). The spectra were extracted and deconvoluted using Data explorer software version 3.4 build 192 (Bruker Daltonics, Bremen, Germany). XPS Analysis XPS analysis was performed using an AXIS Ultra DLD spectrometer (Kratos Analytical Inc., Manchester, UK) with a monochromated Al Kα source at a power of 180 W (15 kV x 12 mA), a hemispherical analyser operating in the fixed analyser transmission mode and the standard aperture (analysis area: 0.3 mm × 0.7 mm) The total pressure in the main vacuum chamber during analysis was typically between 10-9 and 10-8 mbar. Survey spectra were acquired at a pass energy of 160 eV. To obtain more detailed information about chemical structure, oxidation states etc., high resolution spectra were recorded from individual peaks at 20 eV pass energy (yielding a typical peak width for polymers of < 0.9 eV). Samples were filled into shallow wells of a custom-made sample holder and analysed at an emission angle of 0° as measured from the surface normal. Assuming typical values for the electron attenuation length of relevant photoelectrons the XPS analysis depth (from which 95 % of the detected signal originates) ranges between 5 and 10 nm for a flat surface. As the actual emission angle is ill-defined in the case of powders (ranging from 0º to 90º), the sampling depth may range from 0 nm to approx. 10 nm.


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Flow Cytometry Analysis of nanoparticle samples resulting from antifouling, IgG immobilization and protein titration experiments was performed on the BD LSRFortessa™ X-20. A488 and Cy5 fluorescence data was collected using BD FACSDIVA™ software. The collected data was analysed using FLowJo® with gating on the forward scatter and side scatter for doublet discrimination, and graphs produced in Prism (Graphpad Software, USA). In Figure 4A, B we used 1-way-ANOVA to identify statistical differences between surface coatings with 5 % significance level. Multiple comparisons were conducted and significance indicated on graphs by asterisks (**** means p-value ≤ 0.0001, *** ≤ 0.001, ** ≤ 0.01, * ≤ 0.05, ns > 0.05) DLS and zeta potential measurements Dynamic Light Scattering and zeta potential were performed on a Zetasizer Nano ZS with 10 measurements and 3 replicates in Milli-Q, while zeta measurements were conducted using a dip cell with 30 measurements and 3 replicates. The particle size was taken from the Z-average and the results graphed in Prism (Graphpad Software, USA).

Results and Discussion For the library of constructs to be tested (Figure 1), we chose the EK repeat motif for anti-fouling based on previous work by Jiang et al.7 At the C-terminus we included a cysteine for immobilization to surfaces (in this case to form a disulphide with pendant thiol groups on the nanoparticles), we left the N-terminus either unmodified or linked to an IgG-binding Zdomain for oriented antibody binding. As we anticipated that difficulties could encountered in expressing the unnatural peptide sequences, we designed constructs including two different solubility tags (maltose-binding protein – MBP, or thioredoxin – TRX), separated from the peptide by a tobacco etch virus protease (TEVP) recognition site. We also investigated two


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different lengths of EK repeats (EK1 ~ 4 kDa; EK2 ~ 12 kDa). After initial expression and purification (including tag removal experiments), we included a EK2 with a Z-domain construct (EKZ) and evaluated the peptides coated onto organosilica nanoparticles in an ELISA-style sandwich immunoassay for dengue NS1 capture.

We expressed both EK1 and EK2 peptides with and without solubility tags. High level soluble expression was observed for all constructs, with MBP-tagged constructs consistently yielding more product than the un-tagged versions (see Supp Figures S1-S4 for expression analysis of MBP- and TRX-tagged EK1 and EK2 constructs). However, given the propensity for MBP-fusion proteins to precipitate following tag removal, the un-tagged peptides were selected for subsequent production. Supp Figure S5 show typical expression and TEVP cleavage patterns for EK1 and EK2 without any solubility tag. Due to the zwitterionic nature of the peptides, their gel transit patterns were not representative of their predicted molecular weight. However, by comparing against pre-induction samples, peptide expression was clear even after 3hrs under most conditions. Optimal expression was obtained using BL21 Star™(DE3) cells in auto-induction medium at 25C overnight. Following TEVP cleavage, purified EK1 and EK2 were both collected from the flow-through and wash samples, while the elution samples contained different compositions of uncleaved peptide, TEVP, and cleaved His-tag. Purification of EKZ followed a similar process and gel images are included as Supp. Figure S6. The purified peptides were characterised to confirm length and composition (Table 1). The expected molecular weights (ExPASy tool) were confirmed using a combination of mass spectrometry and 1H-NMR for EK1 and EK2 (see Supp. methods for peak assignments), and mass spectrometry for EK1, EK2 and EKZ (Supplementary Figures S7-S9).


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EK peptides were immobilized to thiol-functionalized silica nanoparticles ((3Mercaptopropyl)trimethoxysilane, MPS) , either as single films (MPS@EK1, MPS@EK2 or MPS@EKZ) or as EK1 or EK2 films “backfilled” with EKZ (to form MPS@EK1/Z or MPS@EK2/Z). XPS analysis was employed to confirm changes in surface composition between MPS and MPS@EKx nanoparticles (Table 2). The MPS control samples showed a consistent set of survey and high-resolution spectra to that from previous reports on the same material and so the same peak assignments were used (Figure 2, Supp. Table S1 ).16 In comparison to MPS control samples, all MPS@EKx samples showed attenuation of the Si2p, and S2p signals and increase in C1s and N1s, consistent with a coating having been deposited. In the C1s spectra (Figure 2B), additional peaks corresponding to the C=O/O-CO/N-C=O (287.9 eV) and O-C=O (288.8 eV) were observed and in the N1s (Figure 2C), a double peak assigned to NH3+/(NH2 & HN) (400.8/399.5 eV) were observed all consistent with a peptide coating.8, 17 There was possibly some residual triethylamine catalyst present based on the MPS control samples, yet this was negligible (

Antibody-binding, anti-fouling surface coatings based on recombinant

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