Sortase A-Mediated N-Terminal Modification of Cowpea Chlorotic

Oct 27, 2015 - ... HongDavid N. QuanChen-Yu TsaoWilliam E. BentleyGregory F. Payne ... Lise Schoonen , Sjors Maassen , Roeland J. M. Nolte , and Jan ...
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Sortase A‑Mediated N‑Terminal Modification of Cowpea Chlorotic Mottle Virus for Highly Efficient Cargo Loading Lise Schoonen, Jan Pille, Annika Borrmann, Roeland J. M. Nolte, and Jan C. M. van Hest* Radboud University, Institute for Molecules and Materials, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands S Supporting Information *

ABSTRACT: A new strategy is described for the modification of CCMV for loading of cargoes inside the viral capsid. Sortase A, an enzyme which is present in Gram-positive bacteria, was used to attach cargo to the glycine-tagged N-termini of several CCMV variants. We show that small molecules and proteins bearing a C-terminal LPETG-motif can be attached in this way. This method allows for the site-specific, covalent, and orthogonal modification of CCMV capsids in a mild fashion, leading to high encapsulation efficiencies. This strategy can easily be expanded to other types of cargoes, labeled with an LPETG-tag without altering protein function.

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coupling chemistry.10,12,14 This method is more specific, as it allows cargo to be attached at the desired location. However, this only works for proteins/nanoparticles that do not have a reactive thiol group, limiting the number of applications. Modifications at the N-termini of CCMV proteins are particularly interesting, as these are located on the inside of the VLPs, allowing controlled encapsulation of chemotherapeutics and catalytically active species. Cornelissen et al. used a noncovalent method to attach cargo to the CCMV protein Ntermini, by making use of coiled-coil peptides as linkers.15 However, this method requires targets to be attached to a rather large coiled-coil peptide. Moreover, attaching the target in a covalent fashion to CCMV has been shown to lead to higher encapsulation efficiencies.16 We set out to develop a generic strategy to modify the Ntermini of CCMV proteins with a wide range of different species in a covalent way. This requires a broadly applicable method which can be used to specifically address the Nterminus over other free amine groups. Also, the method should not involve harsh conditions, as these might lead to denaturation/deactivation of the CCMV protein and/or the cargo. Based on these considerations we decided to explore the enzymatic modification of CCMV proteins catalyzed by Sortase A (SrtA). SrtA is present in Gram-positive bacteria, where it is responsible for the anchoring of surface proteins to the bacterial cell wall. Proteins bearing the sorting signal LPXTG (where X = any amino acid) are recognized and the amide bond between Thr and Gly is cleaved. The new Thr C-terminal residue is then coupled to an N-terminal Gly residue of an oligoglycine peptidoglycan crossbridge.17 SrtA has been used for a wide variety of applications, ranging from protein labeling to surface anchoring and engineering of bacterial surfaces.18 Recently,

INTRODUCTION Virus-like particles (VLPs) are protein assemblies, which have appealing features for use in bionanotechnology. They have a well-defined size and shape, are robust and biocompatible, and can be functionalized at both the interior and exterior faces.1−5 The cowpea chlorotic mottle virus (CCMV) capsid is especially interesting in this respect, as it is able to encapsulate relatively large cargoes compared to VLPs derived from other viruses. Also, CCMV capsids can undergo a reversible, pH-dependent assembly and disassembly, even in the absence of their viral RNA.6 The CCMV capsid consists of 180 identical 20 kDa capsid proteins. They form an icosahedral cage with T = 3 symmetry and inner and outer diameters of 18 and 28 nm, respectively.7,8 CCMV VLPs have been modified in many different ways.9 The most popular method for functionalization of the CCMV capsid proteins is by addressing the amino acids which are naturally present on the capsid interior and exterior faces, as this method does not require prior modification of the capsid proteins themselves. Amine and carboxylic acid containing residues have been modified with fluorescent dyes, peptides, haptens, and biotin using N-hydroxysuccinimide (NHS) chemistry,10−12 and carboxylic acid groups have also been used to introduce alkyne groups which could subsequently react with azides using click chemistry via a cycloaddition.13 The disadvantage of addressing endogenous amino acids is the low selectivity of this process: functionalization of a specific residue requires the blockage or modification of residues that should not react with the group of interest. Furthermore, modifications to undesired positions can lead to protein deactivation or denaturation. Cysteines are also often utilized for protein modification purposes. CCMV has two natives cysteines, but these are not susceptible to chemical modification.10 Therefore, surfaceexposed cysteines have been genetically introduced in order to attach cargo using thiol-maleimide or thiol-iodoacetamide © XXXX American Chemical Society

Received: September 5, 2015 Revised: October 23, 2015

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DOI: 10.1021/acs.bioconjchem.5b00485 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Scheme 1. Overview of Sortase A-Mediated N-Terminal Functionalization of CCMV with an LPETG-Labeled Target Molecule

graphy and, if necessary, preparative size exclusion chromatography (SEC), and were subsequently analyzed by polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of the proteins were verified by electrospray ionization time-of-flight (ESI-TOF) mass spectrometry. The modified capsid proteins were assembled to show that they still formed T = 3 particles upon lowering the pH from 7.5 to 5.0. The particles eluted at approximately 10 mL on a Superose 6 column. Transmission electron microscopy (TEM) showed particles with an average diameter consistent with T = 3 shaped VLPs (see Supporting Information for experimental procedures and characterization of expressed proteins). To evaluate the possibility of using the SrtA reaction for the labeling of the capsid proteins with small molecules, FITC-AnlAla-Leu-Pro-Glu-Thr-Gly-NH2 (FITC-LPETG) was employed, containing a FITC-modified azidonorleucine (Anl) amino acid. This reaction could be easily monitored using the fluorescence of the FITC-modified product. The reaction was performed at room temperature, as the capsid protein dimer is unstable upon incubation for prolonged time periods at elevated temperatures. Both substrates were present at a concentration of 50 μM and different amounts of SrtA were added to determine the amount required to achieve high conversions. After addition of SrtA from Staphylococcus aureus to the substrates, the reaction mixtures were incubated for 24 h. SDS-PAGE analysis revealed that higher amounts of SrtA led to more product formation, as expected (Figure 1A and Figure S1). The formation of the

SrtA was used to multifunctionalize protein nanoparticles on their exterior.19 However, until now it has not been explored for the modification of VLPs. Here, we report the modification of the CCMV capsid protein at its N-terminus using a SrtA-based strategy (Scheme 1). We genetically modified the N-terminus of CCMV to display a glycine or triglycine motif. Using SrtA, we were able to covalently attach a peptide and a protein containing a Cterminal LPETG-tag. This method allows for the first time the site-specific, covalent, and orthogonal modification of CCMV VLPs under benign conditions, after expression of the capsid protein. This way, functionalized capsids with a specified amount of cargo inside can be produced after a minimal amount of processing steps. We believe that this strategy can be extended to the attachment of various types of targets, as long as they can be equipped with an LPXTG-tag. Even enzymes incompatible with other coupling methods might be employed to create catalytically active VLPs.

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RESULTS AND DISCUSSION In this study, two types of CCMV were modified: the often used His-tagged CCMV capsid protein and a modified form, containing an N-terminal ELP-tag.20 ELPs consist of repeating VPGXG pentapeptides (where X = any amino acid).21 These thermally responsive peptides can switch from a water-soluble state to a collapsed hydrophobic state by an increase in temperature or salt concentration, which is known as lower critical solution temperature behavior.22−24 The two CCMV types show different assembly behaviors. The regular form of CCMV only shows assembly upon decreasing the pH, i.e., into T = 3 particles. ELP-CCMV on the other hand assembles both via a conventional pH-shift (CCMV-induced assembly) and by an increase of salt concentration or temperature (ELP-induced assembly) into T = 3 or T = 1 particles, respectively.20 To investigate the broad applicability of the coupling reaction for different CCMV forms, we set out to modify the N-termini of both CCMV and ELP-CCMV. The CCMV constructs were designed to display the Sortaserecognition signal at their N-termini. A hexahistidine tag was also included to facilitate purification of the protein by affinity chromatography. In the literature it has been shown that in some cases the SrtA reaction is more effective when two or more glycines are present. However, this highly depends on the accessibility of the N-terminus.25−27 Therefore, two CCMV variants were cloned with one or three glycines to compare the influence of these and the presence of an N-terminal ELP part on the reaction: G-CCMV 1a, G3-CCMV 1b, G-ELP-CCMV 1c, and G3-ELP-CCMV 1d (Scheme 1). The addition of small glycine-tags to CCMV did not affect their soluble expression in E. coli. All proteins were purified by Ni2+ affinity chromato-

Figure 1. SDS-PAGE analysis of Sortase-mediated N-terminal labeling of G-ELP-CCMV 1c with FITC-LPETG. Gels were visualized both by Coomassie blue staining (top) and by in-gel fluorescence (bottom). (A) Conjugation followed over time. (B) Reaction mixtures after 24 h before preparative SEC purification (pre) and purified capsids (post). B

DOI: 10.1021/acs.bioconjchem.5b00485 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 2. (A) SEC chromatogram of G-ELP-CCMV capsids (pink line) and G-ELP-CCMV capsids after modification with FITC-LPETG in the absence (blue line) or presence (orange line) of SrtA. Solid line = absorbance at 280 nm, dashed line = absorbance at 495 nm. (B) Uranyl acetatestained TEM micrograph of FITC-modified G-ELP-CCMV (0.2 equiv SrtA). Scale bar corresponds to 200 nm. (C) Size distribution of the FITCmodified G-ELP-CCMV particles shown in B.

product was particularly visible when the fluorescence of the gel was measured. No conversion to the product was observed when no SrtA was added (Figure S2). Analysis of the reaction mixtures after 24 h by mass spectrometry confirmed the presence of the FITC-G-CCMV and FITC-G3 -CCMV products in the mixtures to which SrtA had been added (Figures S3−S6). In order to compare the four different CCMV variants, quantitative analysis of the Coomassie-stained SDS-PAGE gels was performed. The conversions found for the 0.2 equiv SrtA reaction mixtures were compared, as these showed the least overlap of the SrtA band with the band of the coupled product. After 24 h, the conversions of all proteins were quite similar: CCMV conversions of 53%, 62%, 57%, and 50% were found for 1a, 1b, 1c, and 1d, respectively. Differences between the four CCMV variants were larger after 7 h, as conversions of 37%, 35%, 57%, and 41% were observed for 1a, 1b, 1c, and 1d, respectively. It is known that in the Sortase coupling reaction, an equilibrium is reached. This is due to the fact that a glycine nucleophile is also released upon cleavage of the threonine− glycine bond during the formation of the LPET-SrtA intermediate. This nucleophile can compete with the desired nucleophile. The conversions that were measured in this specific reaction were comparable to examples known from the literature. There are some methods to improve the coupling yield, e.g., the use of depsipeptides or performing the reaction under dialysis.28,29 Luckily, for our application we do not need a high conversion, as we cannot entrap 180 pieces of large cargo inside the CCMV interior. We can conclude from this data that having more N-terminal glycines does not necessarily improve the efficiency of the SrtA reaction in this case. Furthermore, it seems that the modification of 1c is the fastest reaction, as we did not observe an increase in yield anymore after 7 h. Additional advantages of using the ELP-tagged variants over the nonmodified variants are the higher expression yields (∼5−10 times higher) and the ability to use more triggers for capsid assembly besides a pH shift. Therefore, we chose to perform subsequent experiments with G-ELP-CCMV 1c.

Next, it was determined if and to what extent the modified capsid proteins could still be coassembled into VLPs. For this purpose SrtA reactions were carried out for 24 h, and the reaction mixtures were dialyzed to pH 5 buffer to induce capsid formation. The capsids were then isolated by preparative SEC and analyzed by SDS-PAGE (Figure 1B). ImageJ analysis revealed that the FITC-1c:1c ratio in the reaction mixtures was similar to that found in the purified capsids for each SrtA concentration, with a maximum of about 58% incorporated FITC−CCMV. The UV−vis absorbance of the capsids clearly showed that the protein absorbed not only at 280 nm, but also around 495 nm, which is the absorbance maximum of FITC (Figure 2A). Analysis of the capsid proteins by mass spectrometry confirmed the presence of FITC-G-ELP-CCMV (Figure S7). Subsequently, the capsids were analyzed with TEM. TEM micrographs of purified 1c VLPs showed particles with an average size of 29.4 ± 1.7 nm, which corresponds well with that of T = 3 particles (Figure S8). The FITC-modified capsids showed a broader size distribution and a slightly smaller average size of 23.3 ± 2.6 nm (Figure 2B,C). Interestingly, these smaller particles could already be observed after incubation of the capsid proteins and FITC-LPETG without SrtA (Figure S8). We hypothesize that the presence of ELP conjugated to CCMV could be the reason for this effect, as we did not observe this effect for G3-CCMV 1b. Here we found particles of about 28.3 ± 2.5 nm after the reaction with FITC, which corresponds well with T = 3 particles (Figure S8). In order to demonstrate the applicability of this method to larger (bio)molecules, the Sortase reaction was performed between the G-ELP-CCMV 1c and a model protein. For this purpose, a Green Fluorescent Protein (GFP) construct was modified to display the intended LPETG-tag at its C-terminus, as well as a histidine tag for purification purposes (Table S2). GFP-LPETG-H6 was expressed in E. coli and purified by Ni2+ affinity chromatography and preparative SEC. SDS-PAGE analysis showed that the protein had been obtained in pure form. This was further confirmed by ESI-TOF and SEC C

DOI: 10.1021/acs.bioconjchem.5b00485 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry analysis (see Supporting Information for experimental procedures and data concerning the protein expressions). The coupling of GFP to 1c was followed over time and visualized by SDS-PAGE analysis (Figure 3A). The product was

After coupling with GFP, the reaction mixtures were dialyzed to pH 5 buffer and the capsids were isolated using preparative SEC. SDS-PAGE analysis showed that both 1c and GFP-1c were present in the capsids (Figure 3B). Using ImageJ, it was determined that about 10% of the CCMV proteins in the capsids was modified with GFP. For T = 3 particles, this implies that approximately 18 GFP proteins had been encapsulated in the CCMV capsids. The GFP encapsulation could also be quantified using the capsid absorbance at 280 and 395 nm. CCMV capsid proteins absorb at only 280 nm and not at 395 nm, whereas GFP absorbs at both wavelengths. The ratio between the absorbance of the capsids at 280 and 395 nm was used to calculate the GFP to CCMV ratio.15 From the FPLC data in Figure 4, it was calculated that approximately 16 GFP

Figure 3. SDS-PAGE analysis of Sortase-mediated N-terminal labeling of G-ELP-CCMV 1c with GFP-LPETG. (A) Conjugation followed over time. Gel was visualized both by Coomassie blue staining (top) and by in-gel fluorescence (bottom). (B) Reaction mixtures after 24 h before preparative SEC purification (pre) and purified capsids (post). Gel was visualized by silver staining.

Figure 4. SEC chromatogram of G-ELP-CCMV capsids (pink line) and G-ELP-CCMV capsids after modification with GFP-LPETG in the absence (blue line) or presence (orange line) of SrtA. Solid line = absorbance at 280 nm, dashed line = absorbance at 395 nm.

proteins were loaded per capsid (see Supporting Information for the calculations). This corresponds well to the loading that was determined based on analysis of the SDS-PAGE data. Also, this is quite comparable to and even higher than the encapsulation yield of GFP that was achieved using a noncovalent strategy, where up to 15 GFP proteins were encapsulated per capsid.15 With TEM analysis, particles of 26.1 ± 1.7 nm were found (Figure 5).

observed as a band around 50 kDa which appeared slowly over time. A second band around 50 kDa was observed, which possibly corresponds to the SrtA-GFP intermediate. The presence of the product band could be confirmed by labeling CCMV with a DyLight 650 NHS-ester prior to the Sortasemediated coupling. The labeling allowed for the imaging of both 1c and the desired product GFP-1c using in-gel fluorescence in addition to Coomassie staining (Figure 3A). When no SrtA was added, no product was formed, as expected (Figure S9). Evidence for the intended product GFP-ELPCCMV was provided by ESI-TOF (Figure S10). A CCMV conversion of 19% was observed after 24 h when 0.2 equiv SrtA was used as a catalyst. This conversion is relatively low compared to the reaction with FITC, where a conversion of 57% was achieved with 1c under the same reaction conditions (Figure 1A). This is not unexpected, as the conjugation of two proteins is less efficient than the coupling of a small molecule and a protein, due to for instance steric hindrance which limits the accessibility of the reactive moieties. It should be noted that we do not require a high conversion as the capsid interior can only accommodate a limited amount of cargo.

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CONCLUSIONS We have described a method for the site-selective modification of the CCMV capsid interior in a covalent matter, which is the first of this type. SrtA was applied to attach small molecules and proteins, bearing a C-terminal LPETG-tag, to the CCMV protein N-terminus. For small molecules, up to 58% of the CCMV capsid proteins could be modified and capsids were formed with an identical loading. An encapsulation efficiency of 16−18 proteins per capsid was achieved for GFP, which is higher than the one reported for a noncovalent encapsulation strategy of GFP in CCMV capsids.15 Our method was found to be applicable to different types of CCMV, and can likely be extended to all kinds of cargoes, for instance, catalysts or drug molecules. As the SrtA-based strategy is highly selective, it allows for the controlled encapsulation of cargo in CCMV and other VLPs. D

DOI: 10.1021/acs.bioconjchem.5b00485 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

electrophoresis; SEC, Size exclusion chromatography; SrtA, Sortase A; TEM, Transmission electron microscopy; VLPs, Virus like particles

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Figure 5. (A) Uranyl acetate-stained TEM micrograph of GFPmodified G-ELP-CCMV (3.0 equiv SrtA). Scale bar corresponds to 200 nm. (B) Size distribution of the GFP-modified G-ELP-CCMV particles shown in A.

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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.5b00485. Experimental procedures for the cloning, expression, and purification of all proteins, as well as supplementary figures and calculations (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +31 24 36 53204. Fax: +31 24 36 53393. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Jade Heister for the cloning of G3-ELP-CCMV. The Ministry of Education, Culture and Science (Gravitation program 024.001.035) is acknowledged for financial support.

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ABBREVIATIONS CCMV, Cowpea chlorotic mottle virus; ELP, Elastin-like polypeptide; ESI-TOF, Electrospray ionization time-of-flight; FITC, Fluorescein isothiocyanate; GFP, Green fluorescent protein; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel E

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DOI: 10.1021/acs.bioconjchem.5b00485 Bioconjugate Chem. XXXX, XXX, XXX−XXX