General Strategy for Ordered Noncovalent Protein Assembly on Well

Nov 1, 2013 - Jane Besong-Ndika , Matti Wahlsten , Daniela Cardinale , Jan Pille , Jocelyne Walter ... Claudia Koch , Katrin Wabbel , Fabian J. Eber ,...
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General Strategy for Ordered Noncovalent Protein Assembly on Well-Defined Nanoscaffolds Jan Pille,†,‡ Daniela Cardinale,† Noel̈ le Carette,† Carmelo Di Primo,⊥ Jane Besong-Ndika,†,§ Jocelyne Walter,†,# Hervé Lecoq,∥ Mark B. van Eldijk,‡ Ferdinanda C. M. Smits,‡ Sanne Schoffelen,‡ Jan C. M. van Hest,‡ Kristiina Mak̈ inen,§ and Thierry Michon*,† †

UMR 1332 Biologie du Fruit et Pathologie, INRA-Université Bordeaux 2, 71, av. Edouard Bourlaux, CS 20032-33882 Villenave d’Ornon Cedex, France ‡ Institute for Molecules and Materials, Radboud University Nijmegen, Huygens Building, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands § Department of Food and Environmental Sciences, Latokartanonkaari 11, FI-00014 University of Helsinki, Finland ∥ UR 407 pathologie Végétale, INRA, F-84140 Montfavet, France ⊥ INSERM, U869, Laboratoire ARNA, F-33600 Pessac, France # CNRS, Délégation Aquitaine, esplanade des Arts et Métiers, F-33402 Talence Cedex, France S Supporting Information *

ABSTRACT: Here we develop a novel approach allowing the noncovalent assembly of proteins on well-defined nanoscaffolds such as virus particles. The antibody-binding peptide Z33 was genetically fused to the monomeric yellow fluorescent protein and 4-coumarate:CoA-ligase 2. This Z33 “tag” allowed their patterning on the surface of zucchini yellow mosaic virus by means of specific antibodies directed against the coat protein of the virus. The approach was validated by affinity assays and correlative microscopy. The coverage efficiency was ∼87%. Fluorescence and enzymatic activity were fully retained after assembly. The principle of using the combination of a scaffold-specific antibody and Z33-fusion proteins can be extended to a wide variety of proteins/enzymes and antigenic scaffolds to support coupling for creating functional “biochips” with optical or catalytic properties.

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INTRODUCTION Enzymes and protein fluorophores are versatile biomolecules with a broad application potential. In order to be used in biosensory devices, they are often immobilized on a scaffold to allow the sensory event to take place efficiently. For the current biosensors, rather nonselective immobilization strategies are used. The site of immobilization is often not stringently controlled, the biomolecule surface density remains low or there is no molecular positional control during the immobilization process. To develop the next generation of more selective biosensors, the requirement is to improve the biomolecule distribution on various solid supports.1 We have started to derive strategies aiming at using nano-objects as helpers for the positioning of biomolecules. These nanocarriers (NCs) may be considered as intermediate building blocks carrying correctly exposed proteins on their surface. NCs can then be patterned on supports using nanolithography or convective-capillary deposition.2,3 Viruses and virus-like particles are highly interesting natural NCs in this respect; coupling functional molecules to their highly ordered protein backbones is an attractive way to achieve positional control on solid supports. Although these particles are naturally designed for storage and © 2013 American Chemical Society

transport of viral genetic material, they have a good potential as building blocks for a large panel of biotechnology applications. They may be used for enzyme chips, protein selection, molecular therapy, or to study more fundamental problems raised by modern enzymology. (For a review, see ref 4.) Several strategies have been developed to topologically target the covalent grafting of active macromolecules on virus scaffolds. Knowing the structure of virus capsids, genetic engineering can be used to prepare topologically defined ligand grafting. Methionine derivatives bearing azido or alkyl side chains were incorporated in the coat protein (CP) of icosahedral viruses. Utilizing these unnatural amino acids, transferrin was successfully grafted on the surface of these viruses by means of mild copper-catalyzed azide−alkyne cycloaddition. Although the incorporation of these artificial amino acids did not interfere with the capsid assembly, the strategy cannot be generally applied because the subsequent chemical modifications can be deleterious to capsid stability for Received: August 29, 2013 Revised: October 30, 2013 Published: November 1, 2013 4351

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Figure 1. General strategy derived for coupling proteins to virus particles. (a) Interaction of Z33 with human IgG Fc fragment. A molecular model of the Z33-Fc interaction was obtained from the complex between the human Fc fragment (cyan) and fragment B of protein A13 (PDB: 1FC2). The Bdomain was substituted by Z33 (light pink), and an energy minimization was run using Deep View (http://www.expasy.org/spdbv/). The second Fc chain (green) of the IgG constant fragment (PDB: 1FC1) was then added to the model. (b) Scheme of the construct: the Z33 fragment (orange) genetically fused to the protein of interest binds to the Fc of an antibody raised against the virus particle. CH2 and CH3: IgG’s heavy chain constant domains 2 and 3.

some viruses.5 A direct incorporation of the protein of interest coding sequence into the virus genome through a genetic fusion to the CP may appear to be the most direct way to obtain the required functionalized virus particle. Unfortunately, attempts to express large proteins on the surface of eukaryotic viruses have been proven difficult. First, steric hindrance between the resulting bulky protein-CP fusion generally prevents capsid assembly.6 Second, an instability of the genetic construct often results in the excision of inserts in the progeny of the genome,7,8 and third, we also observed that toxic effects of the expressed enzymes in host cells can lead to poor performance.9 Alternative strategies, making use of noncovalent, reversible association between proteins and CP, seem promising. A leucine-zipper-derived peptide was genetically fused to the CP of the cowpea chlorotic mottle virus (CCMV), and its complementary form was fused to the green fluorescent protein (GFP), allowing a reversible coupling through a heterodimeric coiled-coil peptide interaction.10 Although CPs may be considered as programmable capsid units, which can be modified by either genetic engineering or chemical bioconjugation, the modifications can adversely affect the infection cycle in the host or efficiency of the expression system. Moreover, regions involved in the stable supramolecular self-assembly must be left undisturbed. The most promising technological applications of virus scaffolds reside in the diversity of their shapes and physical and chemical properties. However, the limitations discussed above require case-to-case optimizations; general strategies are still lacking. Here we develop a general, versatile approach that is based on three “building blocks”. Virus particles are used as a scaffold. Antibodies raised against the viral CPs serve as the mediator of specific, noncovalent assembly. They coat the virus particle efficiently and facilitate binding of a fusion protein that carries the antibody-binding Z33 peptide tag (Figure 1). The actually used building blocks will be concisely introduced in the following section. Z33 Peptide. The bacterial pathogen, Staphylococcus aureus, produces a 42 kDa factor, protein A (SpA). It contains five homologous immunoglobulin (Ig)-binding domains in tandem, designated domains E, D, A, B, and C. Each SpA domain can bind Fc (the constant region of an Ig) and Fab (the Ig variable fragment responsible for antigen recognition). The Fc binding

site has been localized to the elbow region at the CH2 and CH3 interface of most immunoglobulin G (IgG) subclasses, and this binding property has been extensively used for the labeling and purification of antibodies.11 The Fab specificity involves a site on the variable region of the Ig heavy chain.12 This could weaken the antibody interaction with the virus capsid. The Z-domain, a 59 residue module derived from the SpA B-domain, only binds the constant Fc region with a Kd value of 10−50 nM.14 Starting from the Z domain, a shorter and more stable 33 residue long module (Z33) was selected that binds IgGs with nearly the same affinity as its wild-type counterpart.15 Z33 was previously used for the specific targeting of adenovirus vectors and vault nanoparticles16 to the surface of various cell types including epidermal growth factor receptor (EGFR)-expressing cells17 and gastric18 and biliary cancer cells.19 In this work, we utilize the Z33 peptide for supramolecular assembly of functional biomaterials. This approach enables the decoration of the surface of virtually any virus particle or antigenic scaffold in a simple and efficient way, with almost any protein of interest. Zucchini Yellow Mosaic Virus. As model virus particles, we chose filamentous plant viruses. Compared with tobacco mosaic virus, whose properties have been extensively studied for technological purposes,20−23 the potential of many phytoviruses of various shapes and properties − which can be easily produced in high yield in their host plants − remains to be explored. Among them are flexuous filamentous phytoviruses with a high aspect ratio. The filamentous phytovirus zucchini yellow mosaic virus (ZYMV) has been chosen for this study. ZYMV is a member of the genus Potyvirus. Potyviruses are plant viruses with flexible rod-shaped particles (ca. 750 nm long, 15 nm diameter) packing a single-stranded, polyadenylated, positive-sense genomic RNA. Monomeric Yellow Fluorescent Protein and 4Coumarate:CoA-ligase 2. To demonstrate IgG-mediated decoration of ZYMV particles, we genetically fused the Z33 peptide to two different proteins, resulting in Z33-Proteins (ZPs). Monomeric yellow fluorescent protein (mYFP)24 was chosen for its fluorescent properties. As a model enzyme, 4coumarate:CoA-ligase 2 (4CL2),25 which is involved in the 4352

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cells were grown at 20 °C for 8 to 9 h. The cells were harvested (4000 rpm, 4 °C, 15 min). The pellets were suspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole) and lysozyme was added (1 mg/mL); after 30 min of lysis at 4 °C, the cells were sonicated (Branson Sonicator 250, six bursts of 5 s each interspaced by 5 s cooling, 40% power cycle, power 2). The lysate was clarified by centrifugation at 4 °C (20 000g for 30 min). The proteins were purified by immobilized metal affinity chromatography (IMAC) with Ni2+ immobilized on nitrilotriacetic acid (NiNTA). The proteins of the clarified extract (25−50 mL) were incubated “in batch” with 1 mL of Ni-NTA beads (Qiagen) for 1 h at 4 °C. Beads were packed in an empty column and washed twice with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole). Proteins were eluted in elution buffer (50 mM NaH2PO4, 300 mM NaCl, 500 mM imidazole) and extensively dialyzed in 50 mM phosphate buffer containing 300 mM NaCl, pH 8.00. The purity of the protein was analyzed by SDS-PAGE. 4CL2Z was further purified by size-exclusion chromatography on Superdex 75 10/300 (GE Healthcare) in an Ä KTA Prime system. Protein concentration was determined by measuring absorbance at 280 nm on an Epoch multiplate spectrophotometer (Biotek). Extinction coefficients (ε) were used as determined by the “Protein Calculator v3.3” at http://www.scripps.edu/∼cdputnam/protcalc. html, being 22 450 and 29 330 M−1 cm−1, respectively, for mYFPZ and 4CL2Z. Typical yields were 130 and 5 mg purified proteins per liter culture for mYFPZ and 4CL2Z, respectively. The correct mass and sequence of the proteins was assessed by matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF), size-exclusion chromatography, LC-MS-MS (mass spectrometry), and SDS-PAGE. (See Figures SI 2−6 in the Supporting Information.) Purification of ZYMV. ZYMV purification was done as previously described.29 Infected zucchini squash leaves were harvested 2 to 3 weeks after inoculation and homogenized in 0.3 M potassium phosphate buffer (PB) pH 8.5 containing 0.2% Na-diethyldithiocarbamate and 0.1% mercaptoethanol. The slurry was emulsified with one volume of Freon 113 (1,1,2-trifluoro-1,2,2-trichloroetane). After centrifugation, 1% Triton X-100 was added to the aqueous phase and stirred for 20 min at 4 °C. The virus was recovered by ultracentrifugation, and the pellets were resuspended in 0.02 M PB pH 7.5 and left 6 h at 4 °C with occasional stirring. The suspension was submitted to a slight clarification before the addition of Cs2SO4 to reach a final density of 1.27 g/cm3 and ultracentrifuged overnight. The opalescent virus-containing zone was removed, diluted 10−15 times in 0.02 M PB, clarified, and concentrated by ultracentrifugation. The final pellet was suspended in a small volume of PB. Virus concentration was determined by spectrophotometry (extinction coefficient 0.1% 260 nm = 2.5). Antibody/mYFPZ Affinity Assays. Antibodies used for affinity assays were rabbit-born polyclonal antibodies raised against purified ZYMV particles at INRA Montfavet, France. For a qualitative affinity test, antibodies were mixed with mYFPZ (molar ratio 1:5 and 1:1) and allowed to incubate 5−20 min at room temperature in phosphatebuffered saline (PBS) pH 7.4. Following incubation, the antibodymYFPZ complex was purified by Ni-NTA beads. As a negative control, antibodies without mYFPZ were subjected to the same procedure. Antibody/ZP Titration. The kinetic and equilibrium constants of the interaction were determined by surface plasmon resonance. Experiments were performed at 25 °C with a Biacore T200 apparatus (Biacore, GE Healthcare Life Sciences, Uppsala, Sweden) on CM5 sensor chips (Biacore). α-ZYMV rabbit-born polyclonal antibodies were coupled to the surface in HBS-EP running buffer (Biacore) by reaction of primary amines with N-hydroxysuccinimide, N-ethyl-N(3,3-diethylaminopropyl)-carbodiimide (Biacore amine-coupling kit) until an amount of 1000 resonance units (RUs) was reached. All samples were extensively dialyzed against the running buffer (0.1 M PB, 0.1 M NaCl, 0.05% Tween-20, pH 7.5) before use. The binding experiments were performed using the single cycle kinetics method, which consists of injecting sequentially over the surface increasing concentration of the analyte, in this study ZP, without regeneration step between each sample injection.30 After each binding cycle, the functionalized sensor chip surface was regenerated with a 2 min pulse

biosynthesis of resveratrol, was selected. The resulting fusion proteins are annotated with a superscript Z, hence mYFPZ and 4CL2Z. Both the assembly properties and the protein functionality were successfully characterized.

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MATERIAL AND METHODS

Z33 Amino Acid Sequence. The Z33 amino acid sequence was FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD, as described.15 A bridging sequence encoding GGGGS was fused to the C-terminus to ensure flexibility26 of the Z33 peptide. DNA Constructs pET21a(+)-mYFPZ and pET21a(+)-4CL2Z. pET21a(+)-mYFPZ. For primers used, see SI Table 1. To position the Z33 sequence before the mYFP sequence, two complementary singlestrand oligonucleotides encoding the Z33 sequence flanked by NdeI and BamHI at the 5′ and 3′ ends, respectively, were designed. They were annealed by heating to 95 °C for 10 min in an annealing buffer (20 mM Tris(hydroxymethyl)-aminomethane, 100 mM sodiumchloride, 2 mM ethylenediaminetetraacetic acid, pH 7.5) and slowly cooled to 25 °C. The procedure produced a double-strand DNA fragment with NdeI and BamHI cohesive ends ready for ligation. No further purification was used. The gene encoding mYFP was kindly supplied by the Tsien laboratory (Howard Hughes Medical Institute, University of California San Diego). Standard PCR was used to amplify the mYFP gene with a forward primer including a BamHI site and a reverse primer including a HindIII site. This PCR product was digested with BamHI and HindIII. The pET21a(+) vector (Novagen) was linearized with NdeI and HindIII. The Z33 sequence was ligated into the linearized vector together with the mYFP sequence overnight at 16 °C, forming the pET21a(+)mYFPZ expression vector (product organization: 5′-NdeI-Z33-BamHImYFP-HindIII-3′). Ligation products were cut with EcoRI to digest any uncut pET21a(+) and transformed in an E. coli XL2-Blue (Stratagene) strain. The sequence of the gene was checked through digestion and sequencing (data not shown). pET21a(+)-4CL2Z. For primer used, see Supporting Information Table 2. Vector pQE30−4CL2 containing the 4CL2 gene was used as starting material. The construct of pET21a(+)-4CL2Z was achieved by homologuous recombination in yeast. First, two regions of pET21a(+)-mYFPZ were PCR-amplified to introduce two gaps, one to replace the mYFP coding sequence with 4CL2 sequence and one to insert a DNA fragment containing a yeast 2μ plasmid origin of replication and the yeast TRP1 gene selection marker (Figure SI 1a in the Supporting Information). For recombination, homologous regions were introduced at both sides of the 4CL2 sequence by PCR using primers containing the same 20 bp flap regions as the one ending the two gaps opened in pET21a(+)-mYFPZ (named U1 and L2 in Figure SI 1a in the Supporting Information).The insert was amplified by PCR from the p70S LMV0 K7 (a kind gift from Dr. German-Retana) with primers allowing the insertion of recombining L1 and U2 sequences (Figure SI 1a in the Supporting Information). The four fragments were purified with PCRapace kit (Invitek). Saccharomyces cerevisiae strain YPH501 1 (MATa/MATa ura3-52 lys2-801amber ade2101ochre trp1-Δ63 his3-Δ200 leu2-Δ1) supplied from Agilent was transformed with 0.5 μg of an equimolar mixture of PCR fragments as described.27 All yeast colonies selected for tryptophan auxotrophy complementation on plates containing minimal synthetic-defined base (SD) medium, enriched with all amino acids with the exception of tryptophan, were collected and cultured in liquid SD medium for 24 h at 30 °C. Plasmid DNA was isolated as described28 and transformed into E. coli strain XL1-Blue (Stratagene) by electroporation. The clones were analyzed for the presence of the recombinant plasmid by selective digestion and sequencing (Figure SI 1b in the Supporting Information). Expression and Purification of mYFPZ and 4CL2Z. pET21a(+)mYFPZ and pET21a(+)-4CL2Z were transformed into the E. coli BLR(DE3)-pLysS (Novagen) strain. Typically 1 L of bacteria culture was grown to OD600 0.6 in 2× YT medium containing ampicillin (100 mg/L) and chloramphenicol (50 mg/L). Protein expression was induced by adding 1 mM IPTG (isopropyl β-D-thiogalactoside), and 4353

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(ε333 = 21 000 M−1 cm−1 for p-coumaryl-CoA). Concentrations of enzyme and substrates were adjusted to get a straight line during the first minutes of the reaction. Under these steady-state conditions, the slope was strictly related to the enzyme activity. Typically, the reaction mixture contained 0.5 mM CoA, 0.5 mM p-coumaric acid, 3.125 mM ATP, 3.125 mM MgCl2, and 1.25 mM DTT in 200 μL. Three μg 4CL2Z was added to start the reaction. Kinetic parameters were obtained by fitting a Michaelis−Menten model to an experimental set of initial rates versus substrate concentration using Graphit v. Five pack (http://www.erithacus.com). Supramolecular Assembly in Solution. ZYMV particles were mixed with α-ZYMV antibodies and mYFPZ in a 1:1:5 molar ratio (molar ratio refers to CP number) in 0.1 M PBS pH 8 and incubated for 1 h at room temperature. The samples were dialyzed against a 100× excess of PBS for 96 h, with buffer changes after 2, 5, 22, 26, 30, 46, 50, 56, 70, 74, 78, and 94 h in open microdialysis knobs of 50 μL covered with a cellulose ester dialysis membrane (molecular weight cut off 300 kDa, Spectra/Por Biotech). Controls (Virus + YFPZ and αZYMV with mYFPZ (same ratios)) were used to control for the removal of unbound proteins. For protein quantification, 2, 1, and 0.5 μg of ZYMV CP, α-ZYMV, and mYFPZ were separately subjected to SDS-PAGE in a 12% polyacrylamide gel. The gel was stained with Coomassie InstantBlue (Expedeon), and bands intensity was quantified by measuring peaks area (ImageJ, http://rsbweb.nih.gov/ ij/). These values were plotted as a function of protein quantities. A linear proportionality was observed and the plots were used as a calibration curve to determine the relative amounts of each molecular species in the dialyzed samples (Figure SI 9 in the Supporting Information).

of 20−40 mM NaOH. One flow cell was left blank and used as a reference. Prior to data processing, the recorded sensorgrams were double-referenced using the BiaEvaluation 4.1.1 software.31 The association and dissociation rate constants, kon and koff, respectively, were determined by direct curve fitting of the sensorgrams, as previously described.32 Preliminary observations showed that a significant subset of ZPs did not bind to antibodies. Before binding measurements, calibration-free concentration assays33 were made to determine the concentration of active mYFPZ. This method relies on calculating the ZP concentration by measuring its rate of diffusion from the bulk mobile phase to the sensor chip surface. This involves fitting the observed binding data to a mass-transport-limited 1:1 interaction model with a known value for the mass transport coefficient km and an unknown variable for the ZP concentration. Measurements are made under partially mass-transport-limited conditions. For these experiments, the level of antibodies immobilized on the sensor ship was high (above 10 000 RU). Experiments were run in triplicate at two extreme flow rates (2 and 100 μL/min). The value of km was imposed, and the model was used to find a single concentration value of ZP that best fits the sensorgrams obtained at 2 and 100 μL/min. The value of km depends on D, the diffusion coefficient of the ZP, as follows: ⎛ D ⎞2/3 k m = 0.98⎜ ⎟ 3 ⎝h⎠

⎛ f ⎞ ⎟ ⎜ ⎝ 0.3wl ⎠

(1)

where h, w, and l (height, width, length) depend on the geometry of the flow cell and f is the flow rate. D equals 1.1 × 10−10 m2/s for mYFP.34 A typical experiment is shown in Figure SI 7 in the Supporting Information. Transmission Electron Microscopy (TEM). Carbon-filmed copper electron microscopy grids were coated with a solution of ZYMV (0.3 mg/mL in PBS) then floated on drops of a 5% solution of BSA in PBS and incubated for 1 h. Grids were washed with BSA PBST buffer (PBS, Tween-20 0.05%, BSA 0.1% w/v) and floated on drops of rabbit α-ZYMV polyclonal antibody at different concentrations (0.02 to 0.5 mg/mL) for 1 h. After washing, grids were floated for 1 h on drops of solutions containing various concentrations of mYFPZ or 4CL2Z in BSA PBS-T. These preparations were used with no further treatment for correlative microscopy assays (see below). To probe the presence of ZPs on the particles surface, grids were washed again and incubated by floating on drops of mouse monoclonal α-GFP antibody (1/500 dilution for visualizing mYFPZ interaction) or mouse α-His monoclonal antibody (1/300 dilution for visualizing 4CL2Z interaction) in BSA PBS-T for 1 h. Grids were washed and floated on drops of a solution of α-mouse IgG-gold antibody (diameter of the gold particle 10 nm) in BSA PBS-T (1/20) for 1 h and washed. All incubations were conducted at room temperature. Grids were stained with drops of a 2% w/v solution of phospho-tungstic acid (PTA), pH 7.3. Acquisition was performed on an electron microscopy Philips CM10 FEI. Correlative Microscopy. For correlative microscopy, carboncoated copper transmission electron microscopy finder grids were prepared according to the protocol described above for electron microscopy. To avoid mYFPZ photobleaching, the grid was kept in the dark during the procedure. The grid was placed on a glass slide. One drop of filtered PBS was placed on the grid to keep a thin water layer between the two glass slides. Tape was used to keep a distance of ∼1.5 mm between the glass slides (Figure SI 8 in the Supporting Information). Fluorescence was measured for 12 s between 510 and 560 nm after excitation with wavelengths of 460−500 nm on an epifluorescence E800 Nikon microscope with an HQ2 Cool SNAP CCD detector. The grid was then dried on paper, stained with PTA, and imaged with TEM. Activity of 4CL2Z. Enzyme activity assays were carried out in a buffered solution containing 25 mM NaH2PO4 and 100 mM NaCl (pH 7.5). Stock aliquots of Coenzyme A (10 mM in water) and 10 mM para-coumaric acid in EtOH were stored at −20 °C. 4CL2Z activity (appearance of p-coumaryl-CoA) was followed at 30 °C by monitoring the absorbance increase at 333 nm in the course of time

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RESULTS AND DISCUSSION Engineering of the Z33 Fusion Proteins. The Z33 peptide was used to link the IgG bound to the virus capsid with the protein of interest. Two proteins, monomeric yellow fluorescent protein (mYFP) and an enzyme, 4-coumarate:CoAligase 2 (4CL2), involved in the biosynthesis of resveratrol, a phytoalexin also implicated as a health-promoting compound,35,36 were fused to Z33. The resulting proteins were termed mYFPZ and 4CL2Z. To facilitate Z33 interaction with IgGs, it was positioned at the N-terminus part of the chimeras. The fusion of Z33 to the various proteins of interest was mediated through a short peptide linker (GGGGS) to facilitate Z33 motion.26 A hexa-histidine tag was fused to the C-terminus of the proteins to enable their purification by IMAC (Figure 2). All fusion proteins were found to be soluble in the supernatant of Escherichia coli extracts. mYFPZ was obtained as pure forms after IMAC (130 mg/L culture). The isolation of 4CL2Z (5 mg/L culture) required an additive step of size exclusion chromatography. Molecular weights of mYFPZ and 4CL2Z were assessed by MS and MALDI-ToF (see the Material and Methods and Supporting Information for details) and found to be as expected. The presence of Z33 at the Nterminus did not affect the spectral properties of the fluorescent proteins or 4CL2Z activity (Table 2). This demonstrates the potentialities of Z33 as a robust tag for various antibodymediated interfacing applications. Z33-Tagged Proteins Bind to IgGs. α-ZYMV IgGs and ZPs were mixed at 1 to 1 and 1 to 5 molar ratio and purified by IMAC on Ni2+-beads utilizing the 6XHis tag of the protein of interest (Figure 3). When mYFPZ and α-ZYMV were mixed in a 1:1 molar ratio, mYFPZ did not bind to all antibodies present (Figure 3, center). Considering that one antibody consists of two heavy chains, both with a potential Z33 binding site,13 up to two mYFPZ molecules can possibly bind to one antibody molecule. Therefore, a 1:1 molar ratio can be expected to result in partially unbound IgGs. When mYFPZ was present in five4354

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chip, and recombinant ZPs were tested as analytes for binding. Figure 4 shows one sensorgram (in duplicate) obtained by

Figure 4. Binding of ZPs to IgGs analyzed by SPR. In this experiment, YFPz was injected over the IgGs-functionalized surface sequentially at increasing concentrations (0.063 μM, 0.25 μM, 1 μM). Red traces: two overlaid sensorgrams; black line: best fit to the experimental data (1:1 Langmuir model).

Figure 2. (a) Scheme of the fusion between the Z33 domain and the proteins of interest. (b) SDS-PAGE of the final products, 4CL2Z and mYFPZ. Left lane: protein size marker. Middle lane: purified 4CL2Z with a size of 65 kDa. Right lane: purified mYFPZ with a size of 33 kDa.

injecting sequentially increasing concentrations of ZP. A Langmuir 1:1 model of interaction best fitted these sensorgrams. The rate constants kon and koff were determined using the active concentration of ZP determined by the calibrationfree concentration assay (see the Materials and Methods): kon = 5.7 × 104 ± 0.1 × 104 M−1 s−1, koff = 4.9 × 10−2 ± 0. 1 × 10−2 s−1 and KD = 860 nM. Although the affinity of ZPs for the IgGs remained high (