Engineering Tunable Dual Functional Protein Cage Nanoparticles

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Cite This: Biomacromolecules XXXX, XXX, XXX−XXX

Engineering Tunable Dual Functional Protein Cage Nanoparticles Using Bacterial Superglue Yoonji Bae,† Gwang Joong Kim,‡ Hansol Kim,† Seong Guk Park,† Hyun Suk Jung,‡ and Sebyung Kang*,† †

Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 44919, Korea ‡ Department of Biochemistry, College of Natural Sciences, Kangwon National University, 1, Kangwondaehak-gil, Chuncheon-si, Gangwon-do 24341, Korea S Supporting Information *

ABSTRACT: The selective detection of specific cells of interest and their effective visualization is important but challenging, and fluorescent cell imaging with target-specific probes is commonly used to visualize cell morphology and components and to track cellular processes. Multiple displays of two or more targeting ligands on a polyvalent single template would make it possible to construct versatile multiplex fluorescent cell imaging probes that can visualize two or more target cells individually without the need for a set of individual probes. To achieve this goal, we used encapsulin, a new class of protein cage nanoparticles, as a template and implanted dual targeting capability by presenting two different affibody molecules on a single encapsulin protein cage nanoparticle post-translationally. Encapsulin was self-assembled from 60 identical subunits to form a hollow and symmetric spherical structure with a uniform size. We genetically inserted SpyTag peptides onto the encapsulin surface and prepared various SpyCatcher-fused proteins, such as fluorescent proteins and targeting affibody molecules. We successfully displayed fluorescent proteins and affibody molecules together on a single encapsulin in a mix-and-match manner post-translationally using bacterial superglue, the SpyTag/SpyCatcher ligation system, and demonstrated that these dual functional encapsulins can be used as target-specific fluorescent cell imaging probes. Dual targeting protein cage nanoparticles were further constructed by ligating two different affibody molecules onto the encapsulin surface with fluorescent dyes, and they effectively recognized and bound to two individual targeting cells independently, which could be visualized by selective colors on demand.

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INTRODUCTION Fluorescent cell imaging is commonly used to detect and visualize cell morphology and components and to study cellular behaviors and responses derived from internal and/or external signals within biological systems.1 A wide range of targetspecific fluorescent probes have been developed by combining fluorescent molecules with targeting ligands, including antibodies,2 targeting peptides,3−5 or affibody molecules.6−9 Affibody molecules are genetically engineered antibody mimics that exhibit high specificity and affinity toward their targets and prove the potential for diagnostic applications in biotechnology and therapeutic developments in biomedicine.6,10−14 Because each affibody molecule specifically recognizes and binds to its target cell, a combinatorial display of two or more affibody molecules on polyvalent platforms would allow for dual or multiple targeting capability. Protein cage nanoparticles have been used as attractive polyvalent nanoplatforms for developing nanoscale bioimaging probes and biosensor components because they have a welldefined symmetric hollow shell structure with uniform nanoscale particle sizes.15,16 They are self-assembled from multiple copies of one or a few types of protein subunits in a © XXXX American Chemical Society

precisely controlled manner, and their polyvalent nature often allows uniform multiple small ligands or chemicals to adhere to their surface genetically and/or chemically. Encapsulin (Encap) is a recently developed protein cage nanoparticle isolated from the thermophilic bacteria Thermotoga maritima.17,18 We previously developed Encaps as effective nanocarriers of antigenic peptides and therapeutic and/or diagnostic reagents using protein engineering.19−21 We proved that their genetic and chemical plasticity was amenable to simultaneously introducing various oligopeptides genetically and diagnostic agents chemically at designated positions. Therefore, it is possible to simultaneously impart multiple functionalities, such as targeting and probing functionalities, onto a single Encap. However, genetic fusion of functional proteins onto protein cage nanoparticles often causes a misfolding of functional proteins and/or impairment of the protein cage nanoparticle assembly;22,23 the chemical attachment of functional proteins to protein cage nanoparticles is only limited to very small protein Received: March 15, 2018 Revised: May 17, 2018

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DOI: 10.1021/acs.biomac.8b00457 Biomacromolecules XXXX, XXX, XXX−XXX

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Scheme 1. Schematic Representation of a Multivalent Display of Functional Proteins on Encapsulin Post-Translationally Using Bacterial Glue to Develop Multiplex Fluorescent Cell Imaging Toolkits

inserted between residues 138 and 139 (loop region) of the encapsulin subunit by PCR with pET-30b-based plasmids containing genes encoding encapsulin, which has only one exterior cysteine per subunit at position 123 as a template.19 The amplified DNAs are transformed into competent E. coli strain BL21 (DE3), and the proteins were overexpressed. The E. coli cells containing the resultant proteins were pelleted from 1.0 L of culture and were resuspended in 30 mL of phosphate buffer (50 mM sodium phosphate and 100 mM sodium chloride, pH 6.5). Lysozyme was added, and the solution was incubated for 30 min at 4 °C. The suspension was sonicated for 10 min in 30 s intervals and subsequently centrifuged at 13000g for 1 h at 4 °C. SC-fused monomeric proteins (fluorescent proteins-SC and SCAfb) were purified with immobilized metal affinity chromatography (IMAC, 5 mL HisTrap FF column, GE HealthCare). Encap-L-ST was purified with size exclusion chromatography (SEC) after heat precipitation for 20 min at 65 °C.35 Chemical Conjugation of Fluorescent Dyes to Encap-L-ST. Thiol-reactive fluorescent dyes, such as fluorescein 5-maleimide (Thermo Scientific, 62245), and Alexa fluor 546 C5 maleimide (Thermo Scientific, A10258) were conjugated to Encap-L-ST, which has one outer cysteine per subunit.36 Encap-L-ST was incubated with 5 mol equivalents of fluorescent dyes at room temperature with gentle shaking overnight. Free dyes were removed by dialysis with PBS buffer, and dialysis buffer was changed twice before final overnight dialysis. The degree of fluorescent dye conjugation to Encap-L-ST was determined with UV/vis spectrophotometer and ESI-TOF mass spectrometer. For mass analyses, each fluorescein-conjugated Encap (fEncap-L-ST and aEncap-L-ST) sample was loaded onto a MassPREP microdesalting column (Waters) and eluted with a gradient of 5−95% (v/v) acetonitrile containing 0.1% formic acid at a flow rate of 500 μL/ min. Ligation of SC-Proteins and Encap-L-ST. Concentrations of purified SC-proteins and Encap-L-ST were determined with a UV/vis spectrophotometer and extinction coefficient values of proteins. They were simply mixed with desired molar ratios and incubated for indicated time periods at room temperature with gentle shaking. Reaction resultants were analyzed with SDS-PAGE after adding SDS loading buffer and boiling at 110 °C. Ligations of SC-proteins and Encap-L-ST were further confirmed by measuring molecular masses of monomeric SC-Affibody molecules (SC-HER2Afb and SC-EGFRAfb) and reaction resultants (Encap:HER2Afb and Encap:EGFRAfb) with ESI-TOF mass spectrometer as described above. For characterizing Encap protein cage architecture after ligation of SC-proteins, Encap

partners, and it is difficult to control their number and position.24,25 To circumvent these limitations, we utilized a recently developed bacterial superglue, the SpyTag (ST)/SpyCatcher (SC) protein ligation system.26 Fifteen kDa SC and 13 amino acid ST spontaneously form an irreversible isopeptide covalent bond upon recognition, and they can be genetically fused to any type of protein individually without a significant functional alteration of the fused proteins.27,28 Effective displays of various antigenic proteins on virus-like particles using the ST/SC protein ligation system were recently demonstrated, and their effective inductions of immune responses were successfully shown.29−34 In the present study, we genetically inserted ST to Encap surface loop regions (Encap-L-ST) to generate tunable nanoplatforms and simultaneously displayed multiple types of SC-fused functional proteins, either fluorescent proteins and affibody molecules or two different affibody molecules, in a mixing-and-matching manner (Scheme 1). Using this tunable dual functional Encap, we successfully demonstrated the targetspecific imaging capability of dual functional Encap with multiple combinations of targets and colors as well as the detecting ability of two different target cells with a single dual targeting Encap, which displays two different affibody molecules. The ST/SC protein ligation system and polyvalent Encap made it possible to display multiple types of functional proteins simultaneously on a single platform in various ways on demand and to construct multiplex fluorescent cell imaging probes that can simultaneously detect two or more target cells.

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MATERIALS AND METHODS

Genetic Manipulation and Purification of SC-Fusion Monomeric Proteins and ST-Inserted Encap. SpyCatcher protein with extra amino acids was genetically fused to the C-termini of fluorescent proteins mApple and eYFP (fluorescent proteins-SC) and N-termini of affibody molecules, human epidermal growth factor receptor-2 affibody (HER2Afb), and epidermal growth factor receptor affibody (EGFRAfb) (SC-Afb) using an established polymerase chain reaction (PCR) protocol with pETDuet-based plasmids as a template.27 The residues AHIVMVDAYKPTK with extra amino acids were genetically B

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Figure 1. SDS-PAGE analyses of reaction results of SC-Afb and fEncap-L-ST at various reaction times. Reaction results of (a) SC-HER2Afb and fEncap-L-ST and (b) SC-EGFRAfb and fEncap-L-ST. Reactions were sampled at the indicated times, run on SDS-PAGE, and stained with Coomassie blue. Molecular weight markers were run together, and the apparent molecular weights are indicated (solid arrow indicates SC-Afb molecule-ligated fEncap (fEncap:HER2Afb or FEncap:EGFRAfb). ligated with a single type of SC-protein (Encap:HER2Afb, Encap:EGFRAfb, Encap:mApple, and Encap:eYFP) and Encap with two different types of proteins (Encap:mApple:HER2Afb, Encap:mApple:EGFRAfb, Encap:eYFP:HER2Afb, and Encap:eYFP:EGFRAfb) were measured with size exclusion chromatography (SEC), dynamic light scattering (DLS), and transmission electron microscopy (TEM) analyses with control sample.37 Cell Culture. SK-BR-3 and MCF-7 cells were purchased from the Korean cell line bank (KCLB). MCF-10A and MDA-MB-468 were obtained from American Type Culture Collection (ATCC). SK-BR-3 was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% antibiotic-antimycotic. MCF-10A was cultured in DMEM/F12 (1:1) supplemented with 5% horse serum, 1% penicillin-streptomycin, hEGF (100ug/mL), hydrocortisone (1 mg/mL), and insulin (10 mg/mL). MDA-MB-468 was cultured in Leibovitz’s L-15 medium with 10% (v/ v) FBS, 1% antibiotic-antimycotic, 25 mM HEPES, and NaHCO3. MCF-7 cells were cultured in RPMI-1640 supplemented with 5% FBS and 1% antibiotic-antimycotic. SK-BR-3, MCF-10A, MDA-MB-468, and MCF-7 cells were incubated in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. Fluorescent Cell Microscopic Imaging. The cells (1 × 105/ well) were grown on a microscope cover glass (18 mm Φ) in a 12-well plate (SPL, 30012). The cells were fixed with 4% paraformaldehyde in PBS for 1 h and washed three times with PBS. For nonspecific binding of sample to the background to be prevented, the blocking reagent (5% BSA, 5% FBS, and 0.5% Triton X-100 in PBS) was treated and incubated at room temperature for 1 h prior to sample treatment. After washing fixed cells three times with wash buffer (0.1% Triton X-100 in PBS), SC-ligated Encaps were added and incubated for 1 h at room temperature. Encaps without affibody molecules were also added separately as negative controls under the same conditions. Before sealing, the cells were washed three times with wash buffer, and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images of samples were obtained using an Olympus Fluoview FV1000 fluorescent microscope (Olympus, UOBC).

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they are exposed on the Encap surface, they are accessible to other molecules.19,21 On the other hand, SC is genetically fused to two different affibody molecules, EGFRAfb and HER2Afb, which selectively recognize and bind to epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor2 (HER2) on the surface of specific cancer cells, respectively.27 SC-HER2Afb and SC-EGFRAfb were overexpressed in bacteria and purified with affinity chromatography, and their molecular masses were verified by an electrospray ionization time-of-flight mass spectrometer (ESI-TOF MS). The molecular masses of SC-HER2Afb and SC-EGFRAfb were measured at 21863.5 and 21704.0 Da, which correspond to the calculated molecular masses of each protein, 21864.6 and 21703.6 Da, respectively (Figure S1a and b). Encap-L-ST has an external cysteine per subunit (60 cysteines per cage), and fluorescein-5-maleimide (F5M) was conjugated by a thiol-maleimide reaction and used as a fluorescent probe for fluorescent microscopic cell imaging.38 Fluorescein-conjugated Encap-L-ST (fEncap-L-ST) was examined by a mass spectrometer and a UV/vis spectrophotometer. The molecular mass of the F5M-treated Encap-L-ST subunit was measured at 33831.0 Da, which corresponds well to the sum (33833.2 Da) of the calculated Encap-L-ST subunit molecular masses (33405.8 Da) and F5M (427.4 Da) (Figure S2a). These data suggest that each subunit of the Encap-L-ST was modified with only one F5M without any side reaction. To quantify the molar ratio between the Encap subunit and F5M with an alternate method, we acquired a standard curve from the absorbance of different concentrations of free F5M molecules and measured the absorbance of fEncap-L-ST at 280 and 490 nm, which represented the concentrations of the Encap subunit and F5M, respectively. The obtained molar concentrations translated to ∼98% Encap subunits that were labeled with fluoresceins (Figure S2b), indicating that almost every Encap-L-ST subunit was labeled with fluorescein. Tuning the Polyvalent Display of Affibody Molecules on Fluorescently Labeled Encap Using an ST/SC Protein Ligation System and Exploiting Their Target Specific Binding. To investigate whether genetically fused SCEGFRAfb or SC-HER2Afb can covalently attach to the surface of fEncap-L-ST through an SC/ST isopeptide bond formation, we simply mixed them and sampled at indicated times (Figure 1). Initially, either SC-EGFRAfb or SC-HER2Afb was mixed with fEncap-L-ST with the subunit ratio of 1:2 to consume all of the introduced SC-Afb (SC-EGFRAfb or SC-HER2Afb) and to avoid steric hindrance among ligated affibody molecules.

RESULTS AND DISCUSSION

Construction of ST-Inserted Encap (Encap-L-ST) and SC-Fused Affibody Molecules (SC-Afb). To polyvalently display affibody molecules as targeting ligands onto the Encap surface, we utilized an ST/SC system.26 We genetically inserted a short ST peptide (AHIVMVDAYKPTK) into the surface loop region (L, at the position between subunit residues 138 and 139) of Encap with extra residues (GGGGGASASAS and ASASASGGGGG) on both sides of each ST to provide conformational flexibility and to guarantee full access of their counterparts, SC-fused proteins, and denoted this construct with Encap-L-ST. We previously reported that the loop regions are tolerable for peptide insertion of various lengths, and as C

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Figure 2. Fluorescent microscopic images of various cell lines treated with affibody molecule-ligated fEncap-L-ST, (a) fEncap:HER2Afb and (b) fEncap:EGFRAfb. Cell lines and affibody molecules (with or without) are indicated on the left of the image panels. Nuclei are stained with DAPI (blue, left panels). Fluoresceins are visualized in green (middle panels). Scale bar = 20 μm.

Figure 3. SDS-PAGE analyses of reaction results of SC-proteins and Encap at various reaction times. Reaction results of (a) Encap:mApple:HER2Afb, (b) Encap:eYFP:HER2Afb, (c) Encap:mApple:EGFRAfb, and (d) Encap:eYFP:EGFRAfb. The input ratio of SC-protein to Encap is 2:2:6 [20 fluorescent protein-SC and 20 SC-affibody per Encap-L-ST (60 subunits per cage)]. Reactions were sampled at the indicated times, run on SDS-PAGE, and stained with Coomassie blue. Molecular weight markers were run together, and the apparent molecular weights are indicated (solid arrow indicates fluorescent protein-SC-ligated Encap (Encap:mApple or Encap:eYFP); blank arrow indicated SC-Afb moleculeligated Encap (Encap:HER2Afb or Encap:EGFRAfb)).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of those reactions showed that the ligation between SC-Afb and fEncap-L-ST took almost 6 h to complete (Figure 1a, b), whereas that of monomeric SC-fused and ST-fused proteins was completed almost within 5 min consistent with previous reports.27,39 Previous studies suggested that the reactive aspartic acid of SC needs to be specifically aligned to facilitate ST with SC docking and to complete isopeptide formation. Therefore, we anticipated that the sheet formation of ST in the Encap loop region may be hindered and delayed significantly due to a steric constraint in the loop. To

further confirm isopeptide formation between SC and ST, we performed ESI-TOF mass spectrometric analyses. The molecular masses of the resulting ligation reactions were measured at 55678.0 Da (fEncap:HER2Afb) and 55516.5 Da (fEncap:EGFRAfb), respectively, which corresponded well to the calculated values of 55680.8 Da [21865.6 Da (SCHER2Afb) + 33833.2 Da (fEncap-L-ST)−18 Da (H2O)] and 55517.8 Da [21703.6 Da (SC-EGFRAfb) + 33833.2 Da (fEncap-L-ST)−18 Da (H2O)] (Figure S1). To evaluate the specific binding of each affibody molecule-ligated fEncap toward its target cells, we prepared MDA-MB-468 and SKD

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Figure 4. Fluorescent microscopic images of various cell lines treated with dual functional Encap. (a) Encap:mApple:HER2Afb, (b) Encap:eYFP:HER2Afb, (c) Encap:mApple:EGFRAfb, and (d) Encap:eYFP:EGFRAfb. Cell lines and affibody molecules used are indicated on the left of the image panels. Nuclei are stained with DAPI (blue, left panels). mApple and eYFP are visualized in red and yellow, respectively (middle panels). Scale bar = 20 μm.

ize SC-protein-ligated Encap, we performed size exclusion chromatography (SEC), dynamic light scattering, and transmission electron microscopy (TEM) analyses. Encap ligated with a single protein type (Encap:HER2Afb, Encap:EGFRAfb, Encap:mApple, and Encap:eYFP) or with two different types of proteins (Encap:mApple:HER2Afb, Encap:mApple:EGFRAfb, Encap:eYFP:HER2Afb, and Encap:eYFP:EGFRAfb) were eluted slightly earlier than Encap-L-ST in SEC (Figure S3a), and their hydrodynamic diameters (36.68, 35.26, 39.12, 38.15, 42.26, 40.86, 38.69, and 41.50 nm, respectively) were also correspondingly larger than that of Encap-L-ST (32.59 nm) (Figure S3b). TEM images of negatively stained SC-proteinligated Encap confirmed their intact cage architecture and smudged extra densities at the exterior area, indicating that SCproteins were well-displayed on the Encap surface (Figure S3c). These results indicate that simultaneous ligations of fluorescent protein-SC and SC-Afb onto the surface of Encap-L-ST at defined ratios do not cause any significant alteration to the Encap protein cage architecture. To evaluate the tolerance of ligation amounts of SC-proteins to the surface of Encap-L-ST, we tested three different input ratios of SC-proteins to subunits of Encap-L-ST; 1:1:6 [10 fluorescent protein-SCs and 10 SC-affibody molecules per an Encap-L-ST (60 subunits per cage)] (Figure S4a, d, g, and j, Encap:mApple:HER2Afb, Encap:eYFP:HER2Afb, Encap:mApple:EGFRAfb, and Encap:eYFP:EGFRAfb, respectively), 2:2:6 (Figure S4b, e, h, and k), or 3:3:6 (Figure S4c, f, i, and l) and estimated the degree of covalent conjugations among them with SDS-PAGE (Figure S4). Most of the added SC-proteins were covalently attached to Encap-L-ST. However, reactions of 3:3:6 generated a noticeable amount of protein aggregation, probably due to the steric hindrance of ligated SC-proteins on

BR-3 cells, which overexpressed EGFR and HER2 on their surfaces, respectively. We treated MDA-MB-468 and SK-BR-3 cells with fEncap:EGFRAfb and fEncap:HER2, respectively, and with fEncap-L-ST in parallel as negative controls and monitored them using fluorescent microscopy. Each ligated complex bound to its target cells efficiently (Figure 2a, b, middle panels), whereas fEncap-L-ST without affibody molecules did not bind to either cell line (Figure 2a, b, top panels). Affibody molecule-ligated fEncap also did not bind to MCF-10A or MCF-7 cells, which do not overexpress HER2 and EGFR on their surfaces, respectively (Figure 2a, b, bottom panels). These data indicated that affibody-conjugated Encaps effectively recognize their target cells and selectively bind to them without any significant nonspecific binding visualizing specific target cancer cells. Multiple Functional Proteins were Effectively Displayed on the Surface of Encap by SC/ST Protein Ligation System. We next investigated whether two different functional proteins can be ligated on the surface of Encap-L-ST. We chose fluorescent proteins mApple and eYFP as fluorescent probes instead of F5M. We genetically fused SC to the Ctermini of fluorescent proteins mApple (mApple-SC) and eYFP (eYFP-SC) with extra amino acids as a linker. We previously showed that these proteins can be easily ligated with ST-fused monomeric proteins.27 To display both fluorescent proteins and affibody molecules simultaneously on the surface of Encap, we simply mixed the same amount of fluorescent protein-SC (mApple-SC or eYFPSC) with the SC-Affibody molecule (SC-HER2Afb or SCEGFRAfb) in a mix-and-match manner with Encap-L-ST to form Encap:mApple:HER2Afb, Encap:mApple:EGFRAfb, Encap:eYFP:HER2Afb, and Encap:eYFP:EGFRAfb. To characterE

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Figure 5. Fluorescent microscopic images of various cell lines treated with dual targeting Encap. (a) fEncap:HER2Afb, (b) aEncap:EGFRAfb, (c) aEncap:EGFRAfb, and (d) fEncap:HER2Afb. (e and f) fEncap:HER2Afb:EGFRAfb (top) and aEncap:HER2AFb:EGFRAfb (bottom). Cell lines, affibody molecules, and fluorescein-labeled Encap are indicated on the left of the image panels. Nuclei are stained with DAPI (blue, left panels). Fluoresceins are visualized in green or red (middle panels). Scale bar = 20 μm.

affibody molecules to fluorescent dye-labeled Encap-L-ST. Prior to testing dual targeting, we examined the possibility of cross-targeting among affibody molecule-ligated Encap. We first chemically conjugated Encap-L-ST with either green, F5M (fEncap-L-ST), or red fluorescent dyes, Alexa flour 546 maleimide (aEncap-L-ST). All Encap-L-ST subunits were also labeled with an Alexa fluor 546 maleimide (Figure S5) the same as those of F5M. fEncap-L-ST and aEncap-L-ST were subsequently treated with SC-HER2Afb and SC-EGFRAfb, respectively, to form fEncap:HER2Afb and aEncap:EGFRAfb (Figure S6a). To check the cross-targeting possibility of fEncap:HER2Afb and aEncap:EGFRAfb, we treated SK-BR-3 and MDA-MB-468 cells with fEncap:HER2Afb and aEncap:EGFRAfb (Figure 5a and b), respectively, or aEncap:EGFRAfb and fEncap:HER2Afb (Figure 5c and d), respectively. As we expected, green fluorescence only appeared in fEncap:HER2Afb-treated SK-BR-3 cells, which are targeted by HER2Afb (Figure 5a), and red only appeared in aEncap:EGFRAfb-treated MDA-MB-468 cells, which are targeted by EGFRAfb (Figure 5b). These results imply that each affibody molecule-ligated Encap selectively recognizes its target cells and tightly binds to them without any significant cross-targeting events. To construct a multiplex cell imaging probe that can visualize two or more target cells, we treated fluorescently labeled Encap-L-ST with two different SC-Afbs (SC-HER2Afb and SCEGFRAfb) at the same ratio and the same time (Figure S6b). Encap-L-ST chemically conjugated with one type of fluorescent dye (fEncap or aEncap) was subsequently ligated with both SCHER2Afb and SC-EGFRAfb simultaneously to visualize multiple target cells with a fluorescent probe (fEncap:HER2Afb:EGFRAfb or aEncap:HER2AFb:EGFRAfb). Each target cell was detected by the color that corresponded to the labeled

the Encap surface. To avoid this aggregation issue but maximize functionality, we hereafter used the reaction results of 2:2:6 (Figure 3) for further studies. SEC was used to isolate the dual functional Encaps and remove unreacted monomeric SCproteins prior to further usage. They maintained their integrity and function after the reaction having ∼20 affibody molecules and 20 fluorescent proteins per Encap, which is sufficiently polyvalent for efficient targeting and signal enhancement. Specific Binding of the Dual Functional Encap to the Target Cells. To evaluate the capability of targeted cell imaging of dual functional Encap, we prepared MDA-MA-468 and SK-BR-3 cells and four different combinations of dual functional Encaps (Encap:mApple:HER2Afb, Encap:eYFP:HER2Afb, Encap:eYFP:EGFRAfb, and Encap:eYFP:EGFRAfb) and carried out fluorescent microscopic cell imaging as previously described. Encap:mApple:HER2Afb and Encap:eYFP:HER2Afb selectively bound SK-BR-3 cells were colored red and yellow, respectively. Similarly, dual functional Encaps with SC-EGFRAfb (Encap:mApple:EGFRAfb and Encap:eYFP:EGFRAfb) also selectively bound to their target MDA-MB468 cells with corresponding fluorescent colors (Figure 4), whereas Encap without affibodies did not bind to target cells, consistent with previous results (Figure 2). Furthermore, neither of the dual functional Encaps bound to HER2-negative MCF-7 cells or EGFR-negative MCF-10A cells. These data indicate simultaneous targeting and visualization with dual functional Encap with multiple combinations between desired targeting ligands and desired colors. This approach provides the opportunity for various Encap functionalities by ST/SC protein ligation on demand. Dual Targeting Imaging with Two Different Targeting Ligands. To give dual targeting capability to a single Encap nanoplatform, we further attempted to attach two different F

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Biomacromolecules fluorescent dye (Figure 5e and f) compared with the negative control cell line HEK293T (Figure S7). Together, these results suggest that Encap-L-ST can serve as a multivalent template by efficiently displaying two or more different targeting ligands and can be used to detect two or more target cells simultaneously on demand by a novel protein ligation system, ST/SC. A variety of protein cage nanoparticles have been extensively used as polyvalent nanoplatforms for presenting targeting ligands and functional proteins on their surface. For example, nanobodies, targeting and/or cytotoxic peptides, and influenza virus hemeagglutinin (HA) were genetically fused to ferritins to be displayed on their surface and used for selectively binding their targets or eliciting broadly neutralizing H1N1 antibodies.40−43 Although genetic fusion approach was successful in many cases, each construct should be fully generated whenever the target cells or disease models are changed. In contrast, our approach made it possible to post-translationally decorate a single protein cage nanoparticle with multiple targeting ligands and functional proteins in a mix-and-match manner on demand and may provide new opportunities to develop protein cage nanoparticle-based novel nanoplatforms.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82-52-217-5325. Fax: +82-52-217-5309. E-mail: [email protected]. ORCID

Sebyung Kang: 0000-0001-7394-3550 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (NRF) grant (NRF- 2016R1D1A1B03932580 and 2018R1A6A1A03025810) and Global Ph.D. Fellowship (GPF) program (NRF-2016H1A2A1907554) funded by the Korean government.

CONCLUSIONS In this study, we established a tunable dual functional (targeting and probing or two different targetings) Encap protein cage nanoparticle using bacterial glue, the ST/SC protein ligation system, and demonstrated its target-specific imaging capability with multiple combinations of targeting ligands and colors. ST peptides were polyvalently and uniformly displayed on the Encap surface (Encap-L-ST) through genetic insertion, maintaining its symmetric cage architecture. On the other hand, various types of fluorescent proteins and affibody molecules were genetically fused to SC-protein and prepared independently. SC-fused fluorescent proteins (mApple-SC or eYFP-SC) and affibody molecules (SC-EGFRAfb or SCHER2Afb) were simultaneously displayed on the Encap-L-ST surface via a covalent isopeptide formation between ST and SC, and their combinatorial target-specific fluorescent cell imagings were successfully demonstrated. Dual targeting Encap probes were further established by ligating two SC-fused affibody molecules together on their surface with chemical fluorescent dyes, and they visualized two target cells individually without using a set of individual probes. The polyvalent nature of Encap allowed us to display multiple functional proteins efficiently, and ST/SC bacterial glue made it possible to post-translationally display the desired functional proteins in a mix-and-match manner on demand. The approach we described here can be applied to other protein cage nanoparticles, protein oligomers, protein binding partners, and other functional proteins and may provide new opportunities to develop novel multifunctional delivery nanoplatforms and multifunctional complementary nanoscale building blocks for high-ordered functional nanostructures.

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SC-HER2Afb or SC-EGFRAfb (Figure S1), characterization of fluorescently labeled Encap (fEncap-L-ST or aEncap-L-ST) by ESI-TOF mass spectrometer and UV/ vis spectrophotometer (Figures S2 and S5), dynamic light scattering, SEC profiles, and TEM images of SCprotein-ligated Encap (Figure S3), SDS-PAGE analyses of ligation reaction products (Figures S4 and S6), and fluorescent microscopic images of HEK293T cells treated with the dual targeting Encap (Figure S7) (PDF)

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ABBREVIATIONS Encap, Encapsulin; VLP, virus-like particle; SC, SpyCatcher; ST, SpyTag; Encap-L-ST, SpyTag Inserted Encapsulin; Afb, affibody molecules; EGFRAfb, HER2Afb; ESI-TOF MS, electrospray ionization time-of-flight mass spectrometer; F5M, fluorescein-5-maleimide; fEncap-L-ST, fluorescein 5-maleimideconjugated Encap-L-ST; SEC, size exclusion chromatography; DLS, dynamic light scattering; TEM, transmission electron microscopy; aEncap-L-ST, Alexa flour 546 maleimide-conjugated Encap-L-ST.

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All supporting figures material are available free of charge via the Internet at The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.biomac.8b00457. Molecular mass measurements of two different SC-Afbs and subunits of reaction resultants of fEncap-L-ST and G

DOI: 10.1021/acs.biomac.8b00457 Biomacromolecules XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.biomac.8b00457 Biomacromolecules XXXX, XXX, XXX−XXX