Immobilization and Intracellular Delivery of Circular Proteins by

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Immobilization and Intracellular Delivery of Circular Proteins by Modifying a Genetically Incorporated Unnatural Amino Acid Xiaobao Bi, Juan Yin, Xinya Hemu, Chang Rao, James P. Tam, and Chuan-Fa Liu Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00244 • Publication Date (Web): 05 Jun 2018 Downloaded from http://pubs.acs.org on June 7, 2018

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Bioconjugate Chemistry

Immobilization and Intracellular Delivery of Circular Proteins by Modifying a Genetically Incorporated Unnatural Amino Acid Xiaobao Bi,†# Juan Yin,‡# Xinya Hemu,†Chang Rao,† James P. Tam,*† and Chuan-Fa Liu*† † School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore Supporting Information Placeholder ABSTRACT: Backbone-cyclic proteins are of great scientific and therapeutic interest owing to their higher stability over their linear counterparts. Modification of such cyclic proteins at selected site would further enhance their versatility. Here we report a chemoenzymatic strategy to engineer site-selectively modified cyclic proteins by combining butelase-mediated macrocyclization with the genetic code expansion methodology. Using this strategy, we prepared a cyclic protein which was modified with biotin or a cell-penetrating peptide at a genetically incorporated noncanonical amino acid, making the cyclization-stabilized protein further amenable for site-specific immobilization and intracellular delivery. Our results point to a new avenue to engineering novel cyclic proteins with improved physicochemical and pharmacological properties for potential applications in biotechnology and medicine.

Recent years have seen tremendous progress in the development of precision protein manipulation methods for protein engineering.1-5 These techniques are seeing increasing applications in basic research, materials science, biotechnology and medicine.6-15 Notably, there is a surge of interest in engineering cyclic proteins through head-to-tail backbone cyclization.16-24 A cyclized backbone architecture could improve structural, physical and biological properties when compared to its linear counterpart.18-24 These include advantages of increased stability to proteolysis, heat and chemical denaturation as well as increased biological activity and selectivity.18-24 The applicability of such cyclic proteins could be further enhanced if additional modifications are introduced at predetermined sites. For example, three recent studies have demonstrated that backbone-cyclic proteins modified with synthetic polymers show increased stability, prolonged half-life and improved activity in vivo, properties desirable for protein-based therapeutics.25-27 In one of these studies, Lu et al. reported the preparation of a cyclized version of interferon containing synthetic poly-[Glu(PEG)3]20 for which backbone macrocyclization was effected by sortase A.25 The conjugate showed remarkably improved tumor retention and penetration as well as much prolonged antitumor efficacy. In another study, two orthogonal sortases were used to prepare backbone-cyclic, PEGylated cytokines with improved in vivo activity.26 Both of these studies involve the use of sophisticated procedures whereby the polymer moiety or a chemical handle needed for subsequent polymer attachment must be preinstalled onto a synthetic peptide which is ligated to the protein using a chemical or enzymatic method. In the third study, a polymer-modified circular GFP displayed significantly improved tumor retention.27 Again, sortase was used to backbone-cyclize the protein. A synthetic polymer was then introduced, through in situ

atom transfer radical polymerization (ATRP), near the C-terminus of GFP at a genetically engineered cysteine residue which was prior modified with an ATRP initiator. However, the use of a free Cys for conjugation would be unrealistic if the protein also contains other cysteine residues. We envisioned that a more generally useful strategy to access cyclic proteins carrying specific modifications would be to use the genetic code expansion methodology28-32 which can introduce a non-canonical amino acid at any desired site. The non-canonical amino acid, if carrying a special functional group with unique reactivity, will allow subsequent site-specific protein modification. Such a strategy will also be more straightforward as it involves only two steps starting from the recombinant linear protein: bioconjugation at the genetically incorporated non-canonical amino acid and protein macrocyclization.

Scheme 1. Strategy for chemoenzymatic synthesis of circular proteins containing site-specific chemical modifications via the combination of butelase-mediated cyclization and genetic code expansion technology. Currently, both chemical and enzymatic methods have been developed to prepare backbone-cyclized proteins.33-42 Chemical synthesis provides a versatile tool to generate various circular proteins33-35 but requires refolding of the synthesized proteins to their native bioactive forms, which can be cumbersome for cysteine-rich proteins. In addition, synthesizing proteins of > 100 residues is labour-intensive and technically challenging. Enzymatic methods, mediated by intein, sortase, SpyLigase and butelase 1, offer attractive solutions as they use mild aqueous conditions to accessing circular proteins from recombinant linear precursors in

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their native states.22,36-41 Among these, butelase 1 stands out as the most versatile and robust peptide ligase for protein macrocyclization due to its high catalytic turnover, low µM substrate binding affinity and broad specificity.42-45 Butelase 1 catalyzes transpeptidation after an Asn residue and can accept a diverse range of the incoming amine nucleophiles for new Asn-Xaa peptide bond formation. Hence, our strategy for site-specific modified cyclic proteins entails a novel combination of butelase-mediated macrocyclization with the genetic code expansion methodology. In this strategy, butelase 1 is exploited to mediate protein backbone cyclization in a “near-traceless” manner, while an incorporated unnatural amino acid carrying a tailor-made chemical moiety is used for site-specific protein functionalization with various tags (Scheme 1). Using a model protein, i.e., murine dihydrofolate reductase (mDHFR) – an enzyme that converts dihydrofolate (DHF) into tetrahydrofolate, we show that, after attachment of a biotin moiety or a cell-permeable peptide, the stability-enhanced cyclic mDHFR can be easily immobilized to a solid support or delivered intracellularly with better survivability in living cells. Previous study has showed that p-ethynylphenylalanine can be incorporated into mDHFR at Val44 without significant effect on the enzyme’s activity.46 So the plasmids pETDuet-SI-mDHFRV44TAG and pEVOL-PylT-mbPylRS were cotransformed into E. coli BL21 (DE3) strain for protein expression in the presence of an unnatural amino acid of interest (Figure S1). The plasmid pETDuet-SI-mDHFR-V44TAG carries the SI-dipeptide at the Nterminus of mDHFR as the nucleophile substrate of butelase 1, an amber codon at V44 position for encoding an unnatural amino acid (Uaa), and the NHV motif at C-terminus immediately followed by a His tag for ease of purification. Plasmid pEVOL-PylTmbPylRS encodes the wild type PylRS/tRNACUA pair which can introduce the unnatural amino acid at the amber codon position. mDHFR-UaaV44 was expressed in the E. coli for overnight at 30 °C in the presence of 1 mM ε-Boc-lysine, 1. The resulting mDHFR variant mDHFR-K(Boc)44 was purified with the yield of about 10 mg/L culture medium. ESI-MS data showed an observed mass of 23622 Da (calculated mass 23620.2) (Figure S2), indicating that 1 was incorporated into mDHFR at position 44 with high efficiency and fidelity, and that the first formyl-Met residue was also quantitatively cleaved in situ during protein expression. Next, to test whether mDHFR-K(Boc)44 could be circularized by butelase, 50 µM protein was incubated in the 0.2 M Na-phosphate buffer (pH 6.5) containing 50 nM butelase at 37 °C for 30 min. Then the reaction solution was passed through a Ni-NTA agarose column to remove any remaining linear protein substrate which carries the His tag, because, upon butelase-mediated cyclization, the His tag would have been cleaved from the cyclized protein product. The homogeneity and purity of the cyclic mDHFRK(Boc)44 was confirmed by SDS-PAGE and ES-MS analysis (Figure S3). No protein degradation as a result of attack of internal butelase recognition motifs in the protein was detected, suggesting that these sites are conformation-protected and inaccessible. These data indicated that the combination of butelasemediated cyclization and amber codon suppression technology is a feasible strategy to produce C-to-N cyclic proteins with unnatural amino acids incorporated in the sequence. The unnatural amino acid, if carrying a functional group with specific reactivity, could then further undergo chemoselective reaction for sitespecific protein modification, which offers the possibility to generate new protein products with enhanced functions. So two other lysine derivatives functionalized with an alkene or alkyne group, ε-allyloxycarbonyl (or alloc)-lysine 2 and ε-propargyloxycarbonyl (or praoc)-lysine 3, were also efficiently incorporated into mDHFR at the same position (Figure 1a and S4). Initially, we attempted to use thiol-ene chemistry to conjugate biotin to cyclic mDHFR-K(alloc)44 prepared after butelase-catalyzed cyclization.

The thiol-functionalized biotin labelling reagent H-Cys-LysLys(biotin)-NH2 was prepared using solid phase peptide synthesis (Figure S5). However, only about 30% cyclic mDHFR-K(alloc) could be converted to the desired product after the thiol-ene radical addition reaction (Figure S6). So we used copper-catalysed alkyne-azide cycloaddition (CuAAC) to prepare biotinylated mDHFR. The reaction was completed in 2 h and the protein mDHFR-K(praoc)44 was near quantitatively converted to the desired product, which was confirmed by ESI-MS analysis (Figure 1a). Then butelase was used to cyclize the biotinylated mDHFR in high yield and the purity of cyclic mDHFR containing the biotin moiety was also confirmed by ESI-MS and SDS-PAGE analysis (Figure 1a and S7). Next, we compared the enzymatic activity of biotinylated linear mDHFR and biotinylated cyclic mDHFR. There was no significant difference in catalytic activity at room temperature (Figure 1b). However, cyclic mDHFR had a much higher ability to withstand thermal stress than the two linear forms. It retained most of the enzymatic activity after heating at 55 °C for 1 h whereas the linear forms were almost completely inactivated (Figure 1c). These data show that butelase-mediated cyclization can improve the thermal stability of an enzyme. Moreover, the special modification present in the cyclized enzyme

Figure 1. Characterization of linear and cyclic mDHFR containing site-specific biotinylation via click chemistry and butelasemediated cyclization. (a) ESI-MS analysis of linear mDHFRK(praoc)44 (calcd mass 23602.2 Da, found 23603.0 Da), biotinylated linear mDHFR (calcd mass 24046.7 Da, found 24048.0 Da), and biotinylated cyclic mDHFR (calcd mass 22969.6 Da, found 22969.0 Da). (b) Catalytic activity of linear mDHFR-K(praoc)44, biotinylated linear mDHFR, and biotinylated cyclic mDHFR at room temperature. (c) Catalytic activity of the three mDHFR variants after heating at 55 °C for 1 h. (d) Left panel – scheme for site-specific immobilization of the biotinylated cyclic mDHFR to streptavidin-agarose beads via the biotin-streptavidin linkage. Right panel – enzymatic activity of immobilized enzyme. Black bar: beads only; blue bar: control of non-biotinylated linear DHFR (Activity due to non-specific protein adsorption); red bar: specifically immobilized biotinylated cyclic DHFR. The reduction of dihydrofolate to tetrahydrofolate by DHFR causes a decrease of UV absorbance. Relative DHFR activity was plotted as the OD340 value change (∆OD340) following mixing the enzyme with the substrate.


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Bioconjugate Chemistry provides an attachment site for site-specific immobilization to a solid support. Potential synergic benefits may arise from immobilizing a cyclized enzyme because, while cyclization can improve the thermal stability of an enzyme, immobilization can greatly facilitate the separation of the enzyme from the reaction mixture for recycles and reuse and potentially further increase its stability and pH tolerance.47-49 Moreover, site-specific immobilization can also control the orientation of the protein on the solid support.50 So the biotinylated cyclic mDHFR was subsequently immobilized to streptavidin-functionalized agarose beads. Catalytic activity of the immobilized enzyme was also confirmed by measuring OD340 absorbance change after 20 min reaction (Figure 1d). Therefore, these data confirm that a C-to-N cyclized enzyme was site-specifically immobilized to a solid support

Figure 2. Characterization of TAT-cyclo(mDHFR-HA), TATlinear mDHFR-HA, untagged cyclic mDHFR-HA and linear mDHFR-HA and their intracellular delivery study. (a) ESI-MS analysis of TAT-cyclo(mDHFR-HA) (calcd mass 25473.37 Da, found 25475 Da), TAT-linear mDHFR-HA (calcd mass 26550.49 Da, found 26552 Da), non-conjugated cyclic mDHFR-HA (calcd mass 23609.24 Da, found 23609 Da) and linear mDHFR-HA (calcd mass 24686.36 Da, found 24688 Da). (b) Intracellular delivery and stability of TAT-cyclo(mDHFR-HA), TAT-linear mDHFR-HA, non-conjugated cyclic mDHFR-HA and linear mDHFR-HA as determined by fluorescence microscopy. TATcyclo(mDHFR-HA) and TAT-linear mDHFR-HA was successfully delivered into HeLa cells judged by the colocalization between FITC-antibody labeled proteins (green) and nucleus labeled by DAPI (blue). A decrease in fluorescence signal indicates degradation of the proteins. Scale bars: 50 µm. The development of “smart bullet” protein therapeutics that can target specific tissues or the desired sites within the cells has been gaining tremendous attention in recent years.51 However, the difficulty of protein drugs in passing through the cell membrane and their stability in vivo is always a big concern. Therefore, as another example to demonstrate the potential utility of our strategy, we decided to test whether an intracellularly delivered cyclic protein would have increased stability compared with the linear counter-

part. To this end, we decided to conjugate a cell penetrating peptide (CPP) to the circular protein. TAT peptide, which is derived from the transactivator of transcription (TAT) of human immunodeficiency virus,52 was functionalized with an azide group at its N-terminus and its homogeneity and purity was confirmed by HPLC and mass spectrometry (Figure S8-9). Again, coppermediated click chemistry was used for TAT-protein conjugation. After 1 h reaction, near 90% of the protein was conjugated with the TAT peptide, as confirmed by SDS-PAGE and MS analysis (Figure S10a-c). To prepare the cyclic mDHFR conjugated with TAT, we decided to prepare firstly the cyclic mDHFR-K(praoc)44 and then performed the click chemistry to conjugate TAT to the protein. Similar to the linear mDHFR, the cyclic mDHFR was also efficiently conjugated with the TAT peptide under optimized conditions as confirmed by SDS-PAGE and MS (Figure S10d-e). To monitor the cellular uptake of TAT-conjugated cyclic mDHFR, we prepared a new mDHFR construct which is C-terminally fused to the HA tag followed by the butelase recognition motif and a His-tag. The HA tag would allow easy detection under fluorescence microscopy using an FITC-labelled anti-HA antibody. Protein cyclization and TAT peptide conjugation were performed using the same reaction procedures. TAT-conjugated linear mDHFR-HA was also prepared as a control. The purity of mDHFR-K(praoc)44-HA, cyclic mDHFR-K(praoc)44-HA, TATcyclo(mDHFR-HA) and TAT-linear mDHFR-HA was confirmed by MS (Figure 2a). We envisioned that once the uptaken protein was degraded at its C-terminal part in the cells, the fluorescence signal would be lost due to the disappearance of the HA tag. So the relative intensity of the fluorescence signal can indicate the stability of delivered proteins in cells. So, to assess whether TATcyclo(mDHFR-HA) and TAT-linear mDHFR-HA could be delivered into the cells and to compare their relative stability after cellular uptake, HeLa cells were incubated with TATcyclo(mDHFR-HA) and TAT-linear mDHFR-HA (10 µM) for 1 h. Then the proteins were removed from the cells by washing with PBS and the treated cells were incubated further for 2 h to assess how fast the uptaken proteins would be degraded. The uptake of TAT-cyclo(mDHFR-HA) and TAT-linear mDHFR-HA was also compared with the corresponding non-TAT-conjugated linear and cyclo(mDHFR-HA). After 1 h treatment, intense fluorescence was observed in HeLa cells treated with TAT-cyclo(mDHFR-HA) and TAT-linear mDHFR-HA. Negligible fluorescence was seen in the cells treated with non-TAT-conjugated linear and cyclo(mDHFRHA) (Figure 2b). These data show that TAT successfully delivered its cargo, cyclic or linear mDHFR, into the cells while the non-TAT-conjugated proteins could not be taken up by the cells. Moreover, removal of the proteins from the culture medium led to a decrease in fluorescence intensity in both groups of the treated cells, albeit at different rate. As seen from Figure 2 and S11, at 2 h after protein removal, cells treated with TAT-cyclo(mDHFR-HA) retained a significantly higher level of fluorescence signal than did the cells treated with TAT-linear mDHFR-HA. This indicates that the uptaken TAT-cyclo(mDHFR-HA) had a higher stability in the cells than the TAT-linear mDHFR-HA. Therefore, these results point to the potential of an intracellular targeting CPPcyclic protein conjugate for disease treatment. For example, the so-called enzyme-replacement therapy has been used for the treatment of certain metabolic disorders such as Gaucher’s and Fabry's diseases,53 which involves intracellular delivery of key housekeeping enzymes to counter their deficiency in cells. A more stable therapeutic enzyme that can survive longer in the cell would have improved pharmacological effects. In this study, we have developed a novel strategy to prepare Cto-N cyclic proteins containing site-specific modification by combining the advantages of butelase-mediated cyclization and amber codon suppression methodology. To our knowledge, this is the



first study that provides the “proof of concept” of site-specific immobilization and intracellular delivery of circular proteins with enhanced stability. It has direct implications on the applications of enzymes in industrial biotechnology and medicine. As the fastest known protein ligase, butelase 1 is a “molecular supergluing machine” and a very attractive tool for protein engineering. Sitespecific introduction of unnatural amino acids offers additional ways to modify the circularized proteins with various tags. For instance, modification of such proteins with tissue-specific peptides could lead to new targeted therapies or molecular imaging probes for cancer treatment and diagnosis.54 Combination of these two powerful tools holds great potential to engineer proteins with unusual architectures and with new or enhanced functions. Moreover, our strategy is likely applicable for other peptide ligases such as sortase. In sum, the results obtained from this work point to new approaches of utilizing cyclic proteins for various applications and may inspire new efforts in protein engineering in the near future.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed experimental procedures and Figures S1−S11 (PDF)

AUTHOR INFORMATION Corresponding Author * *

[email protected] [email protected]

ORCID Chuan-Fa Liu: 0000-0001-7433-2081 James P Tam: 0000-0003-4433-198X

Present Addresses ‡ Lee Kong Chian School of Medicine, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore

Author Contributions #These authors contributed equally.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We thank the A*STAR (ETPL-QP-19-06) and Ministry of Education of Singapore (MOE 2016-T3-1-003) and (MOE2012-T3-1001) for financial support.

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Immobilization and Intracellular Delivery of Circular Proteins by Modifying a Genetically Incorporated Unnatural Amino Acid


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Immobilization and Intracellular Delivery of Circular Proteins by

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