SpyCatcher-NTEV: A Circularly Permuted, Disordered SpyCatcher

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SpyCatcher-N : A Circularly Permuted, Disordered SpyCatcher Variant for Less Trace Ligation Xue-Jian Zhang, Xia-Ling Wu, Xiao-Wei Wang, Dong Liu, Shuguang Yang, and Wen-Bin Zhang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00131 • Publication Date (Web): 06 Apr 2018 Downloaded from http://pubs.acs.org on April 7, 2018

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Bioconjugate Chemistry

SpyCatcher-NTEV: A Circularly Permuted, Disordered SpyCatcher Variant for Less Trace Ligation Xue-Jian Zhang, 1,2,§ Xia-Ling Wu,1,§ Xiao-Wei Wang,1 Dong Liu,1 Shuguang Yang,2,* and Wen-Bin Zhang1,* 1

Key Laboratory of Polymer Chemistry & Physics of Ministry of Education, Center

for Soft Matter Science and Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, P. R. China

2

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials,

Center for Advanced Low-dimension Materials, College of Material Science and Engineering, Donghua University, Shanghai 201620, P. R. China

E-mail: [email protected]; [email protected] §

These authors contributed equally to the work.

CORRESPONDING AUTHOR FOOTNOTE: Tel.: + 86 10 6276 6876; Fax: + 86 10 6275 1710; E-mail: [email protected]; Tel.: + 86 21 67874080; Fax: + 86 21 67874077; E-mail: [email protected]

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ABSTRACT. The SpyTag/SpyCatcher reaction has emerged as a powerful way for bioconjugation, but it leaves a folded complex in the product after the formation of the isopeptide bond. To vary the location of the reactive residue and reduce the size of the complex and its potential immunogenicity, we engineer two circularly permuted SpyCatcher variants, SpyCatcher-N and SpyCatcher-NTEV, the latter of which possesses a TEV-recognition site for removal of the fragment containing the catalytic site. Surprisingly, both variants are found to be disordered in solution, yet still retain the ability to form an ordered complex upon reaction with SpyTag with second order rate constants of ~10 M-1· s-1. Cellular expression of a telechelic protein bearing SpyCatcher-NTEV at N-terminus and SpyTag at C-terminus gives both cyclized and chain-extended products. Notably, the monomers exist almost exclusively in the cyclic form owing to its high reactivity in vivo. The fragment containing the catalytic site of SpyCatcher-NTEV can then be removed by TEV digestion, giving a circular protein with minimal trace from the ligation reaction. The plasticity of SpyTag/SpyCatcher reactive pair has promised an ever-expanding toolbox of genetically-encoded peptide-protein reaction with versatile features.

INTRODUCTION Bioconjugation techniques are indispensable for making protein-polymer conjugates and for deriving proteins into diverse topology from their linear precursors.1, 2 Typical methods include those enabled by click chemistry,3 native chemical ligation,4 and traceless chemical ligations,5-9 and those mediated by intein,10 sortase A,11 butelase,12,

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etc. The chemical methods often require highly

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reactive, unnatural functional groups, whose incorporation may reduce protein yields and inactivate proteins. The enzyme-mediated methods are very attractive since they only require a relatively short recognition sequence as the substrate and ligation generates a native peptide backbone with minimal undesired residues. Yet, they have stringent requirement on the location of the substrates and could hardly generate branched structures, which limits their usage for engineering protein topology and making protein-based materials. Genetically encoded peptide-protein reactive pairs, such as SpyTag/SpyCatcher ligation, provide a powerful and versatile way for bioconjugation.14, 15 They are originated from proteins containing spontaneously formed isopeptide bonds such as pilin domains and are completely based on natural amino acids. The high reactivity irrespective of their location in the chain and the stable linkage that it creates lead to a series of applications including the Spy-network hydrogel,16 protein topology engineering,17-20 synthetic vaccine development,21, 22 and synthetic biology.23 This powerful toolbox keeps on expanding, including the supercharged, disordered SpyCatcher(-),24 the orthogonally reactive SpyTag-SpyCatcher mutants25 and the SnoopTag-SnoopCatcher pair,26 and the faster version of SpyTag002-SpyCatcher002.27 Splitting CnaB2-type domains has been proposed to be a general strategy for developing such reactive pairs, as revealed by SdyTag-SdyCatcher28 and other “Tag-Catcher” pairs.29 However, for most of the Catchers, the reactive residue is always near the N-terminus. It would be desirable if the reactive site can be varied to near the middle or the C-terminus of the construct. In addition, the Catchers are usually around 10 kDa which leave a complex of considerable size after ligation. It may affect the properties of the final product in different ways and its potential immunogenicity may limit its application in therapeutics.21, P3


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Recently, Howarth et al developed a SpyLigase-mediated peptide-peptide ligation system via further splitting SpyCatcher into two parts, the SpyLigase and the K-Tag.30 This significantly reduced the residual size from ligation, but the reactivity is compromised and requires the presence of trimethylamine N-oxide to promote the reaction. We envisioned an alternative way to address this problem by finding a SpyCatcher variant which could maintain the high reactivity of SpyCatcher, yet is cleavable by protease after ligation. In this way, the extra component could be removed after ligation by proteolytic digestion. In this contribution, we report two engineered reactive SpyCatcher variants, SpyCatcher-N and SpyCatcher-NTEV, developed by circular permutation of SpyCatcher followed by sequence insertion. Unlike the wild-type SpyCatcher, their reactive lysine is placed near the C-terminus of the construct. The label “N” denotes that the fragment without the reactive lysine is placed at the N-terminus of SpyCatcher. In SpyCatcher-NTEV, a protease recognition site is placed before the strand containing the reactive lysine to make the N-terminal fragment cleavable. Both variants enable efficient ligation both in vitro and in vivo, while SpyCatcher-NTEV further allows the removal of its N-terminal fragment by proteolytic digestion. We demonstrated its utility by the synthesis of a cyclic elastin-like protein and a protein-protein conjugate with minimal trace from ligation.

RESULTS & DISCUSSION Construct Design. In SpyCatcher, the reactive lysine (colored green) is located close to the N-terminus (grey region) and the catalytic glutamic acid (colored blue) is in the second region (purple region) (Figure 1). To enable removal of the second region – the fragment containing the P4


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catalytic site – by proteolytic digestion, the protease recognition site must be programmed into the sequence. It is known that SpyTag and SpyCatcher form a tightly folded, highly stable complex upon reaction, which is often difficult to digest by protease.31 We thus chose to place the proteolytic site at more accessible locations. We envisioned that if circular permutation of SpyCatcher can be successfully performed without losing reactivity, the new linker region between the original first and last strand would be more likely to tolerate to the insertion of recognition sites. Therefore, we first circularly permuted SpyCatcher and added a simple (GGS)3 linker to give the variant SpyCatcher-N (Figure 1). After careful visual inspection of the crystal structure of the SpyTag-SpyCatcher complex,31 the new N- and C-terminus (N’ in Figure 1A) of the SpyCatcher variant was chosen to be in the second loop. It seems to be relatively flexible and solvent exposed, which shall generate the least impact on the stability of the complex (Figure 1A). The topology diagram of the complex is also shown in Figure 1A to illustrate the scheme of circular permutation. Once the reactivity is confirmed, the TEV recognition sequence ENLYFQG is inserted as part of the linker sequence to give a cleavable variant named SpyCatcher-NTEV (Figure 1). It is anticipated that TEV protease would cleave the recognition sequence, leaving only a small peptide tag covalently bonded with SpyTag. The complete sequences can be found in Figure S1 in the supporting information.

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Figure 1. (A) Topology diagram of the secondary structure showing the strategy for circular permutation of SpyCatcher. The SpyTag-SpyCatcher complex is also shown on the right to illustrate the original N- and C- terminus of SpyCatcher and the new N- (N’) and C- (C’) terminus. The SpyTag is colored red, the reactive lysine green and the catalytic glutamic acid blue. The complex structure is generated from the PDB (4MLI) except for the yellow line denoting the inserted region of the new linker. (B) Gene constructs of SpyCatcher, SpyCatcher-N, SpyCatcher-NTEV and SpyCatcher-NTEV-CFP. (C) Amino acid sequence alignment of SpyCatcher, SpyCatcher-N and SpyCatcher-NTEV.

Genes of both constructs were synthesized and separately cloned into pQE-80L vector for expression in Escherichia coli BL21 strains. Proteins were first purified by Ni-NTA affinity P6


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chromatography and then by size exclusion chromatography (SEC). The two SpyCatcher variants were well expressed with yields typically around 50 mg per liter of culture. They are quite stable in solution, showing no signs of aggregation over long term storage at 4 oC. As revealed by SDS-PAGE, both SpyCatcher-N and SpyCatcher-NTEV are pure (Figure 2A). Their identities were further confirmed by MS spectrometry (Figure 2B). The mass spectra were obtained from LC-MS and processed via deconvolution using the MaxEnt1 algorithm in the MassLynx V 4.1 software. The raw mass spectra are shown in Figure S2A. Their SEC profiles were also quite similar to each other, as shown in Figure 2C. However, their retention volumes were considerably smaller than that of the wild-type SpyCatcher, which is quite unusual considering their much lower overall molecular weights (SpyCatcher 15.0 kDa, SpyCatcher-N 11.9 kDa, and SpyCatcher-NTEV 12.5 kDa, see Figure S1 for sequences). It thus suggests that their solution states may be different from that of the wild-type SpyCatcher. The larger retention volume of SpyCatcher implies that it has the most compact structure. By contrast, the significantly lower retention volumes of SpyCatcher-N and SpyCatcher-NTEV suggest that circular permutation may destabilize the structure and make their conformation much more extended and dynamic. To test the reactivity, both variants were reacted with a model telechelic protein based on elastin-like protein bearing SpyTag at the C-terminus (EA where E stands for ELP and A stands for SpyTag).15 The reactions were performed at 4 oC for 24 hours and the products were analyzed by SDS-PAGE (Figure 2A). They both exhibit excellent reactivity in vitro, achieving almost quantitative yields with only slight excess of one of the reactants.

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Solution Structure and Reactivity Profile in vitro. The circular dichroism spectrometry was then used to investigate the solution structure of SpyCatcher-N and SpyCatcher-NTEV. The CD spectra of both SpyCatcher-N (Figure S2B) and SpyCatcher-NTEV (Figure 2D, red dashed line) are characteristic of disordered structure with strong negative peak located at 200 nm. This is in distinct contrast to that of SpyCatcher (Figure 2D, blue dashed line) with the positive signal at 230 nm attributed to the β-turn structure and the strong negative peak at 195 nm. Once the two SpyCatcher variants react with SpyTag , their CD profiles change dramatically. The negative peak shifts to 195 nm and the signal at 230 nm slightly increases (Figures 2D and S2B). It indicates that the secondary structure also changed correspondingly. The CD spectra of the complexes are very similar to that of SpyTag-SpyCatcher complex, indicating that their structures are also quite similar.

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Figure 2. (A) SDS-PAGE analysis of SpyCatcher-N, SpyCatcher-NTEV and their reaction product with EA. (B) Mass spectra of SpyCatcher-N and SpyCatcher-NTEV. (C) SEC overlay of SpyCatcher-N (black), SpyCatcher-NTEV (red) and SpyCatcher (blue). (D) CD spectra of SpyCatcher-NTEV and its reaction product with SpyTag. The spectra of SpyCatcher and the SpyTag-SpyCatcher complex are included for comparison. (E, F) 1H-15N HSQC NMR spectra of SpyCatcher-NTEV (E) and its reaction product with SpyTag (F). The blue square shows the formation of isopeptide bond.

To gain unambiguous evidence about their solution states, we used 1H-15N 2D-HSQC nuclear magnetic resonance (NMR) to investigate the structure of SpyCatcher-NTEV and its reaction product with SpyTag. The spectrum of SpyCatcher-NTEV (Figure 2E) exhibits clustered peaks at the center, indicating that it is mostly unstructured. This is similar to our previous result of SpyCatcher(-).24 After reaction, the spectrum of the SpyTag-SpyCatcher-NTEV complex shows many well-dispersed peaks characteristic of a folded structure (Figure 2F). Notably, the characteristic peak at around δ1H= 6.2 ppm also appeared, as shown in Figure 2F in blue square, representing the formation of isopeptide bond.32 The results reveal that SpyCatcher-NTEV is disordered alone, but forms ordered structure upon reaction with SpyTag. This is a remarkable feature and an example of intrinsically disordered protein possessing autocatalytic activity.24

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Figure 3. (A, B) Yields of the reaction between SpyCatcher-N (A) or SpyCatcher-NTEV (B) and EA in PBS buffer (pH 7.4) for 24 hours at 4 °C, 22 °C, and 37 °C, respectively. (C) Yields of the reaction between SpyCatcher-N (black) or SpyCatcher-NTEV (red) with EA in PBS buffer (pH = 7.4) at 4°C with increasing time. (D) Plot of FRET ratio for the reaction between SpyTag-YFP and SpyCatcher-NTEV-CFP with increasing reaction time. Error bars are standard deviations for triplicate experiments.

We also looked into the differences between the reactivity profiles of SpyCatcher variants and the wild-type SpyCatcher. The most notable difference is perhaps their temperature dependence. While SpyCatcher shows increased yields at higher temperatures,14 both SpyCatcher-N variants have the opposite temperature-dependent reactivity (Figures 3A,B and S3A). The yields can reach more than 95% after 24 hours at 4°C, but drop to merely 20% at 37°C. This is consistent with the disordered structures of SpyCatcher-N variants in solution. It is reasonable that reconstitution between a disordered protein and the peptide tag is promoted better at lower temperatures. At higher P 10


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temperatures, the conformations of SpyCatcher-N variants become much more extended and dynamic, which may hinder their proper reconstitution with SpyTag. For a quantitative comparison, we determined the second order rate constant for the two model reactions with EA at 4 oC. The reaction was followed for both SpyCatcher-N variants at designated time points and the yields were determined by SDS-PAGE and densitometry analysis (Figures 3C, S3B,C). The second-order rate constant were determined to be (11.7±0.5) M-1·s-1 for SpyCatcher-N and (9.2±0.3) M-1·s-1 for SpyCatcher-NTEV (Figure S4). The half-time was 3.2 h for SpyCatcher-N and 3.7 h for SpyCatcher-NTEV. The incorporation of TEV protease recognition sequence in SpyCatcher-N had little effect on their reactivity and kinetic. Compared with SpyCatcher (1.4 × 103 M-1·s-1), both SpyCatcher-N variants have slower kinetics. Nonetheless, the rate constant is still one order of magnitude higher than most common cyclooctyne reagents for their reaction with benzyl azide (~0.96 M-1·s-1).33 In fact, the reaction efficiency is sufficiently high to drive the reaction almost to completion, comparable to most “click” reactions. The reactivity is not affected when fusion proteins are made based on SpyCatcher-N variants. As a proof of concept, we fused SpyCatcher-NTEV to cyan fluorescent protein (CFP) and SpyTag to yellow fluorescent protein (YFP). The equimolar mixture of SpyCatcher-NTEV-CFP and SpyTag-YFP25 gives almost quantitatively the product (Figure S5). The CFP and YFP are a well-known pair exhibiting Förster resonance energy transfer (FRET). The FRET ratio, defined as the ratio of peak intensities (R = IYFP/ICFP), could thus reflect the extent of reconstitution (Figure S6). It thus provides a convenient way to follow the reaction (Figure 3D). A rapid increase of R values was observed, indicating that the two fluorophores were quickly brought together as ligation took P 11



place. The signal plateaued after ~3 hours, which was even faster than the model reaction with EA. It suggests that the reactivity may be further promoted by the neighboring proteins. The high reactivity of SpyCatcher-NTEV in vitro is confirmed again. SpyCatcher-NTEV Enables “Less-Trace” Ligation in vivo. To demonstrate the utility of SpyCatcher-NTEV, we designed a telechelic protein (c-SpyELP), SpyCatcher-NTEV-ELP-SpyTag, containing the reactive sequences at N- and C- termini and ELP in the middle. A linear control (l-SpyELP) was also designed where the reactive aspartic acid (D314) in SpyTag was mutated to alanine to abolish the reaction. The gene constructs of c-SpyELP and l-SpyELP are shown in Figure 4. We envisioned that the expression of c-SpyELP would lead to a circular product in vivo via SpyTag-SpyCatcher-NTEV reaction and the cleavable fragment of the SpyTag-SpyCatcher-NTEV complex could be removed by TEV digestion to give a circular protein (c-ELP) with minimal residues from the ligation reaction (Figure 4).

Figure 4. Cellular synthesis of c-SpyELP and the removal of N-terminal cleavable fragment by TEV digestion. P 12


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The proteins were expressed in 2XYT medium at 16°C for 24 h with IPTG induction. After purification by Ni-NTA affinity chromatography, the crude protein product was first analyzed by SEC (Figure 5A) to separate different fractions which were further characterized by SDS-PAGE (Figure 5B). As shown in Figure 5A and 5B, fraction 1 (F1) with the smallest retention volume is composed mostly of chain-extended oligomers due to intermolecular reactions; fraction 2 (F2) is likely a mixture of the dimer and trimer of c-SpyELP; and fraction 3 (F3) is the major product, the cyclic monomer, with the lowest retention volume in SEC and the smallest molecular weight in SDS-PAGE. The much more compact conformation of c-SpyELP is also evident by its fast mobility in SDS-PAGE and larger retention volume in SEC than the l-SpyELP control, although their molecular weights are essentially the same (Figure 5C). This faster mobility is consistent with previous finding in cyclic proteins, but the SEC profiles are somewhat different. It was found that the linear control of SpyTag’-ELP-SpyCatcher (where SpyTag’ is the unreactive mutant of SpyTag with the reactive aspartic acid mutated to alanine) eluted at essentially the same retention time as the cyclic product due to the pre-organization between the nonreactive SpyTag’ and SpyCatcher via physical complexation.20 In current work, the linear and cyclic polymer elutes quite differently (Figure 5C), indicating that pre-organization is absent. We speculated that when the covalent bond could not form, the destabilized SpyCatcher-NTEV mutant could hardly form a stable physical complex with SpyTag. Judging from the SEC and SDS-PAGE results, it is concluded that the monomeric c-SpyELP exists almost exclusively in the cyclic form, confirming the high reactivity of SpyTag and SpyCatcher-NTEV under cellular environment in vivo. P 13



Figure 5. (A) SEC profile of crude expression product of c-SpyELP. (B) SDS-PAGE analysis of each fraction isolated by SEC. (C) SEC overlay of c-SpyELP and l-SpyELP. (D) SDS analysis of the product, c-ELP, obtained from complete digestion of c-SpyELP by TEV protease. (E) MALDI-TOF mass spectra of c-SpyELP and c-ELP. The calculated mass for c-SpyELP is based on the molecular weight with the loss of one water molecule lost upon cyclization and the N-terminal methionine upon processing.

Efficient Removal of N-Terminal Fragment by TEV Digestion. We then subjected the monomeric product of c-SpyELP to proteolytic digestion by TEV to remove the N-terminal cleavable fragment on the SpyTag-SpyCatcher-NTEV complex (Figure 4). It may be achieved either by expressing TEV protease endogenously after the isopeptide formation or by purified TEV protease in vitro. In current work, it was incubated with TEV protease in 20 mM Tris-HCl buffer (pH = 8.0) at 37 oC for 24 h. The SDS-PAGE analysis shows that the N-terminal fragment on the P 14


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complex are completely removed upon digestion (Figure 5D). This is further confirmed by the MALDI-TOF mass spectra (Figure 5E). The observed molecular weight is attributed to the molecular ion with N-terminal methionine processed and the loss of one water molecule upon cyclization (Mw,calcd. = 31036.8 Da vs. Mw,obsd. = 31046.0 Da). After removal of the N-terminal fragment, the digested product, c-ELP, has a molecular weight of 22226.0 Da, which is close to the expected molecular weight of 22181.9 Da. As TEV protease is highly specific, it further confirms that the proteolytic digestion did occur at the desired site.

Figure 6. (A) Reaction between SpyTag-YFP and SpyCatcher-NTEV-CFP to make protein conjugate and the removal of N-terminal cleavable fragment by TEV digestion. (B) SDS analysis of the

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conjugate of SpyTag-YFP and SpyCatcher-NTEV-CFP and the TEV-digested product. (C) MALDI-TOF mass spectra of the conjugate and the digested conjugate.

The strategy also works for the conjugate of two different proteins. As an example, the conjugate of SpyCatcher-NTEV-CFP and SpyTag-YFP (Figure 6A) was also subject to TEV digestion. As shown in Figure 6B, effective removal of the cleavable fragments of SpyCatcher-NTEV was clearly observed after incubating with TEV protease for 24 hours. The MALDI-TOF mass spectra show a decrease of molecular weight of 9027 Da, which is close to the expected value of the fragment (9004 Da) (Figure 6C). This demonstrate the broad scope of application for SpyTag/SpyCatcher-NTEV reaction beyond protein cyclization. Unique Traits of SpyCatcher-NTEV. It is very impressive that SpyCatcher tolerates extensive engineering including circular permutation, insertion and mutation without losing its chemical reactivity with SpyTag. Although the engineered SpyCatcher variant becomes disordered in solution, its reactivity with SpyTag is well-preserved. The unstructured feature is reminiscent of the new paradigm in protein science, the intrinsically disordered proteins.34, 35 It is remarkable that the lack of a pre-existing stable folded structure does not deprive the proteins’ ability to fold with the cognate peptide tag and form the corresponding isopeptide linkage. We speculated that the covalent bond formation may drastically alter the folding free energy landscape and stabilize the otherwise weak associations. It also contributes to the robustness of SpyCatcher toward engineering. In the absence of the covalent bonding, the folded structure may not be stable, which is evidenced by the big difference in the retention time of c-SpyELP and l-SpyELP in SEC. Previously, it was found that the cyclic ELP and its linear control almost elute around the same time in SEC due to physical P 16


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association of SpyTag mutant and SpyCatcher.20 The significantly smaller retention time of l-SpyELP suggests that similar physical complex is absent in this case (Figure 5C). The SpyTag-SpyCatcher-NTEV complex can be cleaved by TEV protease, both in cyclized proteins and in protein conjugates. It indicates that the linker region is indeed relatively loose and solvent-exposed. Other functional sequences may also be introduced into this region to bring in interesting features such as stimuli-responsive chemical reactivity. CONCLUSIONS In this work, two SpyCatcher variants, SpyCatcher-N and SpyCatcher-NTEV were designed and constructed to allow efficient reaction as well as removal of the N-terminal fragment of SpyCatcher after ligation. Both variants maintain good reactivity in vitro with second order rate constants of ~10 M-1·s-1, which is comparable to most “click” reactions in materials science. Moreover, they are disordered in solution, as revealed by CD and NMR spectroscopy. SpyCatcher-NTEV can thus be considered as a chemically reactive, intrinsically disordered protein. We demonstrate that the SpyTag/SpyCatcher-NTEV reaction is also very efficient in vivo, leading to macrocyclization of ELP in situ during expression. The monomers exist almost exclusively in the cyclic form. The extra part of SpyCatcher-NTEV could be completely removed by TEV protease without affecting the cyclic topology of the protein backbone. The variants add to the ever-expanding toolbox of genetically encoded peptide-protein chemistry with diverse features. This work shall shed light on the rational design and synthesis of various topological proteins with minimal trace from ligation so as to minimize their influence on the structure and properties of the target protein as well as to reduce their potential immunogenicity. P 17



Experimental Section Gene Construction. All oligonucleotide primers were ordered from Invitrogen. Genes encoding SpyCatcher-N and SpyCatcher-NTEV with designed restriction sites were assembled by overlapping PCR and cloned into the bacterial expression vector pQE-80L (Qiagen Inc.) by standard restriction digestion and ligation protocols. The full sequences were confirmed by direct sequencing. Protein Synthesis, Purification and Characterization. Plasmids containing the foreign genes were used to transform chemically competent Escherichia coli strain BL21. Unless noted otherwise, the expression was performed in LB or 2xYT broth containing 100 µg/mL ampicillin at 37 °C for 5 hours with isopropyl-β-D-1-thiogalactopyranoside (IPTG) induction at a final concentration of 1 mM/L. The cell lysis and protein purification on Ni-NTA slurry under native conditions were carried out according to standard protocols using single point elution (Lysis buffer: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH=8.0; Wash buffer: 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH=8.0; Elution buffer: 50 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole, pH=8.0 ). After purification, the proteins were buffer-exchanged into PBS (pH = 7.4). Size exclusion chromatography was performed on a Superdex 200 Increase 10/300 GL column in an ÄKTA FPLC system (GE Healthcare, Inc.) using PBS (pH = 7.4) as the mobile phase at a flow rate of 0.5 mL·min-1. The SDS-PAGE was performed on 12.5% or 15% gels to analyze the proteins. Before loading, the samples were mixed with 5x SDS-PAGE loading buffer (250 mM Tris-HCl, 50% glycerol, 10% SDS, 250 mM β-mercaptoethanol, 0.05% bromophenol blue) and heated at 98 °C for 10 min.

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Instrumentations. Ultra-performance liquid chromatography-electrospray ionization mass spectrometry (UPLC-ESIMS) analyses were performed on a system equipped with an ACQUITY H-Class UPLC (Waters Corp.) and a quadrupole rods SQ Detector 2 mass spectrometer (Waters Corp.). A protein BEH C4 column (Waters 300Å, 1.7µm; 2.1×50 mm) was used for separation with ultrapure water (with 0.1% formic acid) and acetonitrile as the mobile phase. The spectra is processed from the raw m/z spectrum via deconvolution using the MaxEnt1 algorithm in the MassLynx V 4.1 software (Waters Corp.). Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was conducted on a MALDI TOF/TOF 5800 mass spectrometer (AB Sciex, USA) with sinapinic acid as the matrix. CD spectra was recorded by a Chirascan-plus spectrometer (Applied Photophisics Ltd., United Kindom) using a 10 mm cuvette. Samples were scanned every 1 nm from 190 nm-260 nm with 0.1 mm path length for 3 repeats. The collection time per point was set as 0.5 s. 1

H-15N HSQC NMR Spectroscopy. The

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N-labelled SpyCatcher-NTEV was expressed in M9

medium (6.8 g Na2HPO4, 3.0 g KH2PO4, 0.5 g NaCl, 0.49 g MgSO4·7 H2O, 1 g

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NH4Cl and 1 L

dd-H2O) and purified with the same procedure as described in previous section. The samples for NMR spectroscopy were prepared in PBS buffer (pH = 7.4), containing 10% D2O. Reactions were peformed at 4 oC with unlabelled SpyTag (sequence: AHIVMVDAYKPTKGSGS). The NMR experiments were performed at 15 °C on a Bruker-700 UltraShield spectrometer (Bruker Corp.), equipped with a CryoProbe. All spectra were processed and analyzed with MestReNova. Reactivity Assay. All reaction assay was carried out in PBS buffer (pH = 7.4). The reactivity profile was obtained by reacting EA and SpyCatcher-N (or SpyCatcher-NTEV) at 4 oC for 24 hours. P 19



The molar ratio between EA and SpyCatcher-N (or SpyCatcher-NTEV) is 1:1.1(1:1.2) and the concentration of EA is 20 µM. The temperature dependence experiments were carried out under identical conditions at 4, 22 and 37 oC, respectively. The time course experiments were performed with equimolar mixture of EA and SpyCatcher-N (or SpyCatcher-NTEV) at a concentration of 20 µM each over a period of 24 h at 4 oC in PBS buffer. Aliquots were taken at different time points and boiled with denaturing buffer to quench the reaction for SDS-PAGE analysis. For SpyTag-YFP and SpyCatcher-NTEV-CFP reactivity assay, the experiments were carried out under identical conditions at 4 oC for 24 h with [SpyTag-YFP]:[SpyCatcher-NTEV-CFP] = 9.5 µM:8.0 µM. For kinetic experements by FRET, the reaction was performed with [SpyTag-YFP]:[SpyCatcher-NTEV-CFP] = 3.0 µM:7.8 µM and the fluorescence signal at 527 nm was tracked periodically upon excitation at 436 nm at 4 oC

using EnSpire multiplate reader (PerkinElmer Inc.).

TEV Protease Cleavage. Protein sample in PBS buffer (50 mM) was mixed with TEV protease solution at a final molar ratio of 3:1 in a 20mM Tris-HCl buffer (pH = 8.0) and incubated at 37 oC for 24 h. The 5x SDS-PAGE loading buffer was used to quench the reaction for analysis. ACKNOWLEDGMENTS We are grateful for the financial supported from the National Natural Science Foundation of China (Grants 21474003, 91427304), the 863 Program (2015AA020941), and “1000 Plan (Youth)”.

NOTES The authors declare no competing ﬁnancial interest.

ASSOCIATED CONTENT

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Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI:XXXXX. Amino acid sequences, SDS-PAGE images, other characterization data, and additional experimental details (PDF)

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SpyCatcher-NTEV: A Circularly Permuted, Disordered SpyCatcher

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