Abstract
The precise modification or functionalization of the protein C-terminus is essential but full of challenges. Herein, a chemical approach to modify the C-terminus is developed by fusing a cysteine protease domain on the C-terminus of the protein of interest, which could achieve the non-enzymatic C-terminal functionalization by InsP6-triggered cysteine protease domain self-cleavage. This method demonstrates a highly efficient way to achieve protein C-terminal functionalization and is compatible with a wide range of amine-containing molecules and proteins. Additionally, a reversible C-terminal de-functionalization is found by incubating the C-terminal modified proteins with cysteine protease domain and InsP6, providing a tool for protein functionalization and de-functionalization. Last, various applications of protein C-terminal functionalization are provided in this work, as demonstrated by the site-specific assembly of nanobody drug conjugates, the construction of a bifunctional antibody, the C-terminal fluorescent labeling, and the C-terminal transpeptidation and glycosylation.
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Introduction
Chemical modification of proteins has emerged as a versatile strategy for the gain of functions, such as increasing the half-life1, labeling the target receptor2, modulating protein-protein interactions3, etc. Great efforts have been made to site-specifically modify the proteins of interest (POIs)4,5, including N- or C-terminal modification6,7, the side-chain of amino acid8, such as amine (lysine)9, sulfhydryl (cysteine)10,11, imidazole (histidine)12,13 and phenol (tyrosine)14. Despite this, site-specific functionalization of proteins, particularly for C-terminus, remains a great challenge suffering from the multiple reactive sites on proteins. Recently, chemo-enzymatic C-terminal modification of the POIs has been widely studied7,15. This involves fusing the C-terminus with unique tags that can be “decorated” by the corresponding enzymes, such as sortase A16,17,18, trypsiligase19, tubulin tyrosine ligase (TTL)20, asparaginyl endopeptidases (AEPs)21,22,23,24,25,26, PAM1527, carboxypeptidase Y (CPaseY)28, and the combination of multiple enzymes29. The chemical approach for C-terminal modification is based on the difference in oxidation potential between terminal carboxylic acids and in-chain Glu or Asp residues with a decarboxylative photoredox methodology30. In addition, the split intein system has been well-developed in the past two decades, especially by Tom W. Muir31,32,33, to achieve the semi-synthesis or N/C-terminal modification of proteins, and has become an important chemical tool to study protein functions. The intein domain can self-split or be removed by MESNA (sodium 2-sulfanylethanesulfonate) and give a thioester intermediate which could further react with an N-Cys-containing protein/peptide or small molecule. Besides, Thom et al. employed the intein system to achieve hydrazinolysis of POI C-terminus by simply incubating POI-intein with hydrazide and then following up with a chemoselective reaction with ketone or aldehyde containing moieties34. Meng-Jung Chiang et al. also constructed an Fc Domain protein-small molecule conjugate by fusing intein onto the C-terminus of Fc, which was converted into an Fc-thioester by MESNA and reacted with a small molecule containing N-Cys-linker35. Another similar approach was developed by Qiao et al. that the POI is fused with a Cys on the C-terminus and is activated by 2-nitro-5-thiocyanatobenzoic acid (NTCB), which generates an intermediate that can be attacked by a subsequent additional nucleophilic molecule36. Collectively, in contrast to numerous strategies for modifying the N-terminus or side-chain of amino acids, the chemical methods for C-terminal functionalization are scanty and limited. Meanwhile, the well-established C-terminal modification approaches are mainly either enzyme-dependent or only suitable for transpeptidation, which creates a difficult scenario (Supplementary Fig. 1).
The cysteine protease domain (CPD) is one of the core elements of the multifunctional automatic processing repeats in toxin (MARTX) toxin37,38,39, which has been widely used as a C-terminal tag to improve the expression, solubility or purification of POIs40,41,42. Compared with other tags applied in protein expression, removing the CPD tag is quite convenient by only a catalytic equivalent of InsP641, a commercially available small molecule. As we found, blocking the free cysteine of CPD will inhibit the self-releasing process, indicating that the free cysteine is the key residue for trigging the self-cleavage43. Accordingly, we conjectured that a thioester intermediate formed during the process of InsP6-induced CPD cleavage. Thioester, which can further react with hydrazine, amine, cysteine, etc., is an important intermediate in the Native Chemical Ligation (NCL)44. Herein, we found that the C-terminus of the POIs could be synchronously modified by an amine-containing molecule during the InsP6-triggered CPD self-cleavage. Examples of specific C-terminal functionalization of POIs with various substrates were also provided, along with their uses in several diverse biological applications, such as nanobody-sugar conjugate, nanobody-drug conjugate, biparatopic antibody, C-terminal fluorescent labeling, etc.
Results
Identification of CPD-induced C-terminal functionalization
CPD, essentially, is a cysteine protease that could be activated by the small molecule InsP6. As discussed above, we found that the modification of the sole free cysteine of CPD will inactive its protease activity, demonstrating that the cleavage of peptide bond is Cys dependent43. Normally, the mercapto group of cysteine attacks the peptide bond and forms a thioester intermediate which could be further hydrolyzed by water to form a free carboxyl group. In addition, the thioester intermediate is a preferable substrate to react with amine-containing molecules. More recently, Rehm et al. reported the thioester intermediate during [C247A]OaAEP1-catalyzed aminolysis25. Hence, we proposed that the InsP6-induced CPD self-cleavage undergoes the thioester intermediate as well, which could be amidated by amines during hydrolysis, and then achievies the C-terminal modification and functionalization of POIs.
To test our hypothesis, we incubated a CPD fusion protein, Endo-F3(D165A)-CPD40,43 (100 μM), with 3-azidopropylamine (50 mM) in PBS at 4 °C for 30 min with or without (w/o) InsP6 (100 μM) (Fig. 1). The reaction was analyzed by LC-MS to check the molecular formula of Endo-F3(D165A). According to the LC-MS results, incubation of Endo-F3(D165A)-CPD (molecular weight 55616) with InsP6 gave two major LC-MS results (Fig. 1a), 23745 (consistent to CPD) and 31888 (consistent to Endo-F3(D165A)). Meanwhile, incubation of the fusion protein only with 3-azidopropylamine would not result in any molecular weight change of the fusion protein (Fig. 1b), indicating that InsP6 could release CPD from POI but not an amine molecule. However, as shown in Fig. 1c, incubating the fusion protein with 3-azidopropylamine and InsP6 gave a molecular weight of 31970, which equals 31888 (M.W. of Endo-F3(D165A)) + 100 (M.W. of 3-azidopropylamine) – 18 (M.W. of water), and a molecular weight of 23745 (M.W. of CPD). To confirm that the amidation occurs during CPD self-cleavage, we further performed a control experiment in which the fusion protein is first incubated with InsP6 for 2 h and then 3-azidoprpylamine is added. As expected, the LC-MS analysis result (Fig. 1d) is consistent with the one without 3-azidopropylamine (Fig. 1a), demonstrating that the released CPD will not transfer the amine group onto the C-terminus but during the self-cleavage process.
Condition optimization of the C-terminal modification
Next, we sought to optimize the reaction conditions. First, the model substrate Endo-F3(D165A)-CPD (100 μM) was incubated with 3-azidopropylamine (2–50 mM) in the presence of InsP6 (100 μM) in PBS at 4 °C. The reaction was monitored by LC-MS (Fig. 2) and the percentage of ligation product or hydrolysis product was summarized in Table 1. We found that 20–50 mM of 3-azidopropylamine will complete the reaction within 2 h and give a 100% ligation product (Fig. 2; Table 1, entry 1-2). However, when the amine concentration was lowered to 5 mM, 2 h’s incubation gave a yield of 85%, but a 15% hydrolysis product was found (Fig. 2; Table 1, entry 3). Further lowering the amine concentration to 2 mM gave only 30% aminolysis product and 70% hydrolysis product respectively (Fig. 2; Table 1, entry 4). From these results, we could observe that the CPD-induced C-terminal aminolysis is a concentration-dependent reaction, indicating a competitive reaction between the amine and water molecules to the thioester intermediates. Additionally, we found that the percentage of hydrolysate increases in a time-dependent manner (Fig. 2; Supplementary Fig. 2; Table 1, entry 3–5), which suggests a reversible reaction that the C-terminal modification could be again released by CPD and give a free C-terminus of the POI.
InsP6 binds to a conserved basic cleft that is distant from the protease active site, and the binding induces an allosteric switch that leads to the autoprocessing and release of CPD in a concentration-dependent manner37,38. Hence, we hypothesized that a lower InsP6-to-substrate ratio may weaken the hydrolysis, and give a better balance between hydrolysis and aminolysis. The evaluation results showed that lowering the InsP6 concentration (500 to 5 μM) will significantly promote the aminolysis yield from 62 to 90% (Fig. 3a, Table 1, entry 4 and 6–10), which is consistent with our speculation. However, both the aminolysis and hydrolysis were inhibited when further decreased InsP6 to 1 μM or 0.2 μM (Fig. 3a, Table 1, entry 11–12). Finally, 5 μM of InsP6 was chosen for further optimization to avoid significant hydrolysis, and the aminolysis product could remain stable in the reaction system for a relatively longer time, which is convenient for subsequent analysis and purification (Supplementary Fig. 3).
Additionally, we performed a pH scan to identify the suitable pH range for CPD-induced C-terminal aminolysis. We chose sodium phosphate buffer (PB) and set the pH at 6.0, 7.0, 8.0, and 9.0 as the final pH value (Supplementary Fig. 4). As shown in Fig. 3b and Table 1 (lane 13–16), we found that the reaction gives 100% hydrolysis when the final pH is <= 7.0, and gives higher aminolysis yield as pH rises. Considering the instability of POIs under strong alkaline conditions, we ultimately chose pH 8.0 as the ideal pH. Further, we also assessed the influence of buffer systems on the CPD-mediated C-terminal modification. As illustrated in Fig. 3c, despite that the working pH range of NaOAc buffer is 3.6–5.8, the CPD-induced C-terminal modification in NaOAc buffer (pH 8.0, lane 22) performs best, giving the ligation products in 94% yield at 5 h, and 88% at 16 h, an odd phenomenon hard to be explained. However, the employment of HEPES, Tris, MOPs, Tricine, or PB buffer gave a relatively unsatisfied yield (Table 1, entry 17–21, Supplementary Fig. 5). Since some buffer solutions are amine-based, such as Tris, HEPES, and Tricine, we tested if any aminolysis occurred by performing the InsP6-triggered CPD self-cleavage of Endo-F3(D165A)-CPD in these buffers, as a negative control. All of the LC-MS monitoring results are identical and equal to the molecular weight of Endo-F3(D165A), proving that these buffers are compatible with the current reaction (Supplementary Fig. 6).
Subsequently, we investigated if a catalyst can facilitate the CPD-mediated C-terminal modification. MESNA, Thiophenol, 4-mercaptophenylacetic acid (MPAA) that are extensively used in NCLs, as well as N-hydroxybenzotrizole (HOBt), 4-dimethylaminopyridine (DMAP), and 4-pyrrolidinopyridine (PPY) which are frequently used in accelerating acylation reactions, were used for the assessment. First, the ability of these chosen catalysts (5 mM) to accelerate the reaction rate was tested. As indicated in Fig. 4a, a decrease in conversion was observed when MESNA, Thiophenol, or MPAA was used, which is presumably owing to the reversible interaction of these substrates’ free thiols with the POI-CPD thioester intermediate. Notably, DMAP and PPY exhibited the ability to promote aminolysis reaction, gave an amidation yield of 76 and 70% respectively, and DMAP showed the best catalytic activity (Supplementary Fig. 7-8). As we know, DMAP and its derivative PPY are strong catalysts for acylation, and our results also indicated that they possess high nucleophilicity towards thioester as well. The formed “acyl-DMAP cation” is a more labile intermediate than thioester to amines, hence improving the reaction rate and yield. Further, we investigated the influence of DMAP concentration. According to the LC-MS results, the aminolysis products predominated when the DMAP concentration rose, while lowering the total reaction efficiency (Fig. 4b, Supplementary Fig. 9). Meanwhile, 5 mM or 10 mM of DMAP showed similar catalytic activity. Hence, 5 mM of DMAP was used, as the catalyst, for further studies. In addition, we also found that CPD-induced C-terminal modification could occur at 4 or 25 °C, indicating a good temperature tolerance (Fig. 4c, Supplementary Fig. 10).
Free CPD could serve as an independent enzyme to mediate C-terminal functionalization and de-functionalization
As discussed above, we observed a reversible reaction that the C-terminal modification will be again released from the product and give a free C-terminus (with carboxyl) when the reaction time is prolonged, indicating that the released CPD is catalytic in the presence of InsP6. To test our hypothesis that free CPD can also act as an independent enzyme to catalyze the amidation, we incubated the isolated aminolysis product, Endo-F3(D165A)-Az, with InsP6 and the purified CPD at room temperature for 12 h. As expected, the LC-MS results (Supplementary Fig. 11a) indicated the release of the azide group from the protein, giving a molecular weight of 31888 that is consistent with the Endo-F3(D165A) with a free C-terminus. However, whether Endo-F3(D165A)-Az was incubated separately with InsP6 or with CPD, the azide group remained on the C-terminus (Supplementary Fig. 11a). Additionally, we blocked the free thiol group of Endo-F3(D165A)-CPD with maleimide-modified peptide to prevent the self-cleavage. And further incubations of the caged POI-CPD were performed individually with InsP6, InsP6 + CPD, or InsP6 + native Endo-F3(D165A)-CPD. According to the LC-MS results, incubation with the InsP6 failed to remove the Cys-blocked CPD from the fusion protein. But, incubation with either InsP6 + CPD or InsP6 + Endo-F3(D165A)-CPD did release the Cys-blocked CPD from the fusion protein, suggesting that the combination of InsP6 and CPD may act as a tool to accomplish the chemoenzymatic protein modification (Supplementary Fig. 11b).
CPD-induced C-terminal modification is compatible with diverse amine substrates and proteins
With the optimized conditions, we then examined the substrate compatibility of this method with various amine-containing molecules. First, we replaced 3-azidopropylamine with other primary-amine molecules, such as propargylamine, ethylenediamine, 5-aminopentan-1-ol, 1-amino-2-methylpropan-2-ol, hydrazide, etc., to react with Endo-F3(D165A)-CPD in the presence of InsP6 and DMAP. The LC-MS analysis suggested a good conversion of the fusion protein to C-terminal functionalized Endo-F3(D165A) with these substrates (Fig. 5a, Supplementary Fig. 12). We also found that PEGylated molecules and labeling tags, biotin for instance, are compatible with the method, but not the secondary-amine molecules (Supplementary Fig. 13a). In addition, the reaction capability was also assessed with different amino acids. However, only glycine can be conjugated onto the C-terminus (Fig. 5a, Supplementary Fig. 13b). Further testing with the dipeptide Gly-Gly or tripeptide Gly-Gly-Gly revealed the successful conjugation of these peptides onto the C-terminus at a favorable yield (Fig. 5a).
Motivated by these results, we turned to fuse the CPD onto more proteins and explore the possibility of CPD-induced C-terminal functionalization. First, diverse POIs with a CPD motif fused to their C-terminus (POI-CPD) were expressed, including a nanobody against Her2 receptor, GFP, Endo-A, Endo-S2, etc. These POIs were successfully constructed and purified from E.Coli system (Supplementary Fig. 14). The obtained fusion proteins (100 μM) were incubated with 3-azidopropylamine (5 mM or w/o), InsP6 (5 μM), DMAP (5 mM), respectively. As illustrated in Fig. 5b, c, the fused CPD could be released from these fusion proteins in the presence of InsP6, and the addition of 3-azidopropylamine resulted in the successful and highly efficient C-terminal azidation of the proteins (Fig. 5c, Supplementary Fig. 15-19), demonstrating the universality of this approach.
Functionalization of target proteins
Compared to the conventional antibody, nanobody possesses unique features, like small molecular weight, high stability, better solubility, and low immunogenicity, which endues nanobody with good tumor penetration ability and easy manufacture. Bioconjugation of a nanobody with functional groups such as fluorophore, peptide, and toxin can improve the efficacy and potency of nanobody and expand its applications in cancer treatment and diagnosis. Therefore, with the established C-terminal functionalization strategy, we next aimed to modify the Her2 nanobody C-terminus for the gain of functions.
In our study, we found that the expression of native Her2 nanobody in E.Coli system is scarce. In contrast, the fusion of CPD onto the Nb C-terminus significantly increased the expression yield (from 1-2 mg/L to 200 mg/L). First, under optimal conditions, Her2 Nb-CPD was rapidly hydrazinolyzed by incubating with hydrazine and InsP6. According to the LC-MS, Nb-hydrazide was quantitively obtained without hydrolysis (Fig. 6a, Supplementary Fig. 20a). With Her2 Nb-hydrazide in hand, we introduced a GlcNAc onto the C-terminus in an approximately 86% conversion by incubating Nb-hydrazide and GlcNAc in 100 mM NaOAc buffer, pH 4.0 (Fig. 6a, Supplementary Fig. 20b). To test the potential in NCL, we treated the Nb-hydrazide with NaNO2 and MPAA (mercaptophenylacetic acid) to form the intermediate Nb-MPAA thioester, which then reacted with cysteine and the Flag tag with N-Cys. According to the LC-MS results, the Cys or Cys-containing peptide was successfully ligated onto the C-terminus of Her2 nanobody (Fig. 6b, Supplementary Fig. 20c-d).
At present, Nanobody-Drug Conjugates (NDC) is a booming research field. With Nb-Az in hand, we incubated it with the linker-payload of BCN-Lys(PEG24)-VC-PAB-MMAE45 (BCN, bicyclo[6.1.0]nonyne; PEG, poly(ethylene glycol); VC-PAB, Valine-Citrulline-para-aminobenzyl alcohol; MMAE: monomethyl auristatin E, a cytotoxin) to afford an NDC that conjugates drug MMAE on the C-terminus (Fig. 7a, Supplementary Fig. 21a-b). In another application, we also synthesized a nanobody-FITC (fluorescein isothiocyanate isomer I, a fluorescence) probe by reacting the Nb-Az with DBCO-FITC (DBCO: dibenzocyclooctyne) (Fig. 7b, Supplementary Fig. 21c). Since the molecular weight of the Her2 Nb is around 13 kDa, we also used RP-HPLC to identify the modification process. From the HPLC results, we could clearly figure out that either the aminolysis or functionalization step has a good yield (yield > 95%, Fig. 7c). The excess linker-drug/FITC could be simply removed from the reaction mixture by centrifugation, and the pure Her2 NDC or Nb-FITC was efficiently achieved. After that, the in vitro anti-tumor activity of the NDC was evaluated against the Her2-positive cancer cells, SK-Br-3, and Her2-negative cell line MDA-MB-231 as control. As shown in Fig. 7d, e, the NDC, compared to the naked nanobody, exhibited potent and selective anti-tumor activity against the Her2-positive cell line. Meanwhile, the fluorescent SDS-PAGE indicated the successful conjugation of FITC onto the Nb (Fig. 7f). The obtained Nb-FITC was then used in confocal microscopy determination, which could bind to the surface of SK-Br-3 cells but not MDA-MB-231 cells according to the results (Fig. 7g). These findings suggested that the conjugation of functional groups onto the C-terminus will not compromise the target protein’s biological functions, suggesting the potential for exploiting protein functionalization by the CPD-induced C-terminus modification.
Next, we covalently linked the Her2 Nb onto trastuzumab to construct a biparatopic antibody. Previously, we reported the traceless site-specific labeling of native antibodies on Fc K248 (EU numbering) using ligand-directed modification27. Here, we synthesized a DBCO- or MTz-tagged trastuzumab by incubating trastuzumab with FcBP-TE-PEG4-DBCO (Supplementary Fig. 23; FcBP, Fc binding peptide; TE, thioester) or FcBP-TE-MTz (Supplementary Fig. 24; MTz, methyl tetrazine) respectively, which (5 mg/mL, 33.4 μM) was further incubated with the Her2 Nb-Az (133 μM, 4 eq) or Nb-TCO (Supplementary Fig. 22, 500 μM, 15 eq; TCO, trans-cyclooctene) at 37 °C for 8 h (Fig. 7h). The reaction mixtures were analyzed by SDS-PAGE. A new band at ~ 70 kDa, which corresponds to the molecular weight of trastuzumab heavy chain plus nanobody, was observed (Fig. 7i, Supplementary Fig. 25), indicating the successful formation of the biparatopic antibody. According to the results, the conjugation of Trastuzumab with Her2-Nb by TCO-MTz pair is more efficient than the one by DBCO-Az pair, with a yield of 86 vs 32% respectively, which provides a feasible approach for chemically constructing bifunctional antibodies.
Discussion
The chemical C-terminal modification of the protein of interest has long been a challenge because the carboxylic acid group is quite ordinary and is hard to selectively react with the C-terminus. Here, by employing a self-cleavage tag, we developed a facile chemical approach for protein C-terminal modification and functionalization with diverse amine-containing substrates. CPD tag is widely used to increase protein expression. It can be released from the protein in a self-catalytic approach that is triggered by InsP6. We found that during the CPD self-cleaving step, the thioester intermediate will react with the amine molecules and form a new amide bond. In this paper, a systemic reaction optimization of the CPD-mediated C-terminal modification was studied, including InsP6 concentration, buffers, pH, and the catalysts. Besides, a reversible reaction was found that the presence of CPD and InsP6 will further hydrolysis the C-terminal modification, enabling a de-functionalization process. Further, we proved that this approach has a wide compatibility of the amine substrates and can functionalize diverse proteins, such as endoglycosidases, GFP, and the nanobody. By this method, a hydrazide-modified nanobody was efficiently obtained which was ulteriorly functionalized with a GlcNAc or a Flag tag via NCL. Additionally, an azide-tagged nanobody was facilely synthesized via this strategy and was employed to construct nanobody-sugar/drug conjugates, nanobody-fluorescence conjugates, as well as the biparatopic antibody.
As we discussed in the introduction, numerous strategies have been developed to modify the C-terminus of POIs, and lots of them are widely used in the functionalization of target proteins, such as Sortase A, AEPs, etc46. However, the existing methods are majorly chemoenzymatic approaches which not only need the expression and purification or isolation of the enzymes but also the design and expression of POIs with fixed C-terminal tags that can be recognized by their unique enzymes. In addition, the donors for modification are majorly amino-acids-based. In other words, these approaches are transpeptidase-catalyzed reactions (Supplementary Fig. 1). Our report, in another aspect, provides a chemical, enzyme-free C-terminal modifying system. In this method, we could achieve C-terminal functionalization by simply incubating POIs-CPD with amines and InsP6, a chemical trigger, making the modification more convenient and avoiding the expression of additional enzymes. Moreover, the presented method is compatible with amine-containing substrates rather than univocal amino acids (to date, only OaAEP1 was reported that it could serve as a tool to modify the C-terminus with diverse amines25,26). Meanwhile, the CPD tag can increase the soluble expression of proteins that are challenging to express, thus providing feasible solutions to broad POIs and applications. For instance, Endo-F3 is a powerful endoglycosidase, but the expression in E.Coli system resulted in low yields (<1 mg/L) of soluble enzyme due to the formation of insoluble inclusion bodies, an awkward situation solved by infusing CPD onto the C-terminus (give a yield of > 15 mg/L)40. In our report, the CPD tag also raised the expression yield of Her2 nanobody in 100–200 folds (from 1-2 mg/L to 200 mg/L). However, though CPD is currently an effective tag for the expression of non-soluble proteins, the co-expressed CPD (211 amino acids) is a huge domain rather than a small tag, which is a potential problematic for unknown proteins. In addition, the released CPD can continue its activity in hydrolyzing the modified POIs to give a free C-terminus residue, another weakness we need to avoid, a similar phenomenon occurred on PALs but solved by a series of following studies47,48,49,50,51,52.
In summary, this approach directly introduces functional tags onto the C-terminus of proteins, enabling rapid product functionalization and diversification in a chemical means. It also implies the possibility of CPD as an efficient protein engineering tool showing a latent application prospect in biotechnology and medicine research, such as nanobody/antibody-drug conjugates.
Methods
Materials
The pET26b-SDAB-Her2 nanobody vector and pCMV6-AC-GFP vector was kindly provided by Professor Xiaohua Chen and Associate Professor Sulin Zhang from Shanghai Institute of Materia Medica, respectively. pGEX5-GST, pCW-Endo A, pET26b-Endo-S2, and pET22b-EndoF3(D165A)-CPD vectors were synthesized by GENEWIZ (Suzhou, China). All designed primers were synthesized by Sangon Biotech (Shanghai, China). Trans5α and BL21 (DE3) chemical competent cells were purchased from Tsingke (Beijing, China). Other materials used in expression and purification were purchased from Sangon Biotech (Shanghai, China). Sodium phytate (InsP6), propargylamine, glycyl-glycine, glycyl-glycyl-glycine, biotin, pyrrolidinopyridine (PPY), 4-mercaptophenylacetic acid (MPAA), FITC, and 2-(tritylthio)acetic acid were purchased from bidepharm (Shanghai, China). 4-dimethylaminopyridine (DMAP), 3-azidopropylamine, sodium 2-sulfanylethanesulfonate (MESNA), N-hydroxybenzotrizole (HOBt), and 2-aminoethanethiol were purchased from J&K Chemical Ltd (Shanghai, China). Glycine, cysteine, and other amino acids were purchased from GL Biochem (China, Shanghai). Fc binding peptide was purchased from Genscript Biotech Corporation (Nanjing, China). DBCO-CONHS and BCN-O-PNP were purchased from Flechem (Shanghai, China). NH2-PEG4-TCO was purchased from Confluore Biotech (Xi’an, China), and MMAE was purchased from Resuperpharmtech (Shanghai, China). Trastuzumab (cat. BDS15-20210301) was ordered and produced by TOT Pharma (Suzhou, China). Centrifugal filtration tube was purchased from Millipore Corporation. Other chemical reagents and solvents, unless indicated, were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). Nuclear magnetic resonance (NMR) spectra were measured on a Varian-MERCURY Plus-400 or 500 instrument.
Cell culture
SK-Br-3 (TCHu225) and MDA-MB-231 (TCHu227) cell line was obtained from Cell Bank of Chinese Academy of Sciences (Shanghai, China), which were grown in RPMI-1640 medium supplemented with Penicillin (50 units/mL), Streptomycin (50 μg/mL) and 10% FBS (fetal bovine serum), and were incubated in a water-saturated cell incubator (Thermo Scientific) at 37 °C under 5% CO2.
High-performance liquid chromatography (HPLC)
Analytical RP-HPLC analysis of protein modification was performed on a Thermo Ultimate 3000 instrument with a PIRP-S column (Agilent, 8 μm, 1000 Å) at 70 °C. Mobile phase A: water containing 0.25% TFA; mobile phase B: acetonitrile containing 0.25% TFA. The column was eluted with a linear gradient of 0–100% B in 25 min at a flow rate of 1.0 mL/min.
Liquid chromatography and electron spray ionization mass spectrometry (LC-ESI-MS)
The LC-ESI-MS data of protein samples were performed and collected on a Waters Xevo G2-XS Q-TOF with a Waters C4 column (ACQUITY UPLC Protein BEH C4, 1.7 μm, 2.1 mm×50 mm) with at 80 °C. Mobile phase A: water containing 0.1% formic acid; mobile phase B: acetonitrile containing 0.1% formic acid; Elution gradient 0-2 min, 5-5% B; 2–10 min, 5–90% B; flow rate: 0.3 mL/min; detecting absorbance: 280 nm.
SDS-PAGE
Proteins were mixed with 1x protein loading buffer (Reducing or No Deturation & Reducing Buffer, YEASEN) and boiled at 100 °C for 5 min before separation on 12% SDS-PAGE gels. In-gel fluorescence was detected, which was followed by Coomassie staining and imaging on a Bio-Red Chemi Moc MP.
Cell viability assay
Her2 positive tumor cells SK-Br-3 and Her2 negative tumor cells MDA-MB-231 were seeded in 96-well plates at approximately 6000 cells/well in 90 μL of growth media and incubated overnight at 37 °C in a 5% CO2 cell incubator. Each sample was diluted 5 folds with RPMI 1640 medium from an initial concentration of 0.5 mM, total of 9 concentration gradients were tested. 10 μL of each concentration was added into 3 repeat wells, and the 96-well plates were cultured in a cell incubator with 5% CO2 at 37 °C for three days. Then 10 μL of MTT solution (5.0 mg/mL in PBS) was added and incubated at 37 °C for 4 h, which was followed by 90 μL of 10% SDS solution to dissolve the formazan overnight. Optical density (OD) values were measured at 570 nm using a BioTek Epoch. The EC50 values and the cell viability curve were calculated by GraphPad Prism software.
Cell staining and imaging on a laser scanning confocal microscope
SK-Br-3 cells or MDA-MB-231 cells were seeded in an 8-well chamber with 60,000 cells/well in 200 μL of RPMI-1640 medium, and the plate was incubated at 37 °C with 5% CO2 for 12 h. The cells were washed with wash buffer (PBS containing 5% FBS) 3 times. 200 μL of Her2 Nanobody-FITC (10 μg/mL in wash buffer) was added to the wells and the plate was incubated for another 1 h at 4 °C, then was washed 3 times with wash buffer. The cell nuclei were stained with Hoechst 33258 (Yeasen, 40729ES10, 1: 1000 dilutions, 100 μL) at room temperature for 30 min. All the cells were washed and preserved in the wash buffer on ice at dark. Image analysis was performed on a Leica TCS-SP8 STED instrument. Hoechst 33258 was excited at 405 nm and the nuclei were detected within a window of 415–460 nm. FITC was excited at 488 nm and was detected with a window of 498–551 nm. Blank cells without any pretreatment but stained with Hoechst 33258 were first analyzed while adjusting the HV/gain/offset parameters to give clear nuclei images and an invisible FITC background. Cells from the experimental group was then analyzed with the same parameters as blank cells.
Homologous recombination of the plasmids expressing various CPD-tagged proteins
A seamless cloning reaction was applied to construct the plasmids expressing CPD-tagged proteins according to the manufacturer’s procedures. First, the pET22b plasmid encoding CPD was amplified from pET22b vector encoding EndoF3(D165A)-CPD (forward primer: 5’-GTCGACGCATTAGCGG ATGGAAAAATACTCCATAA-3’ and reverse primer: 5’-ATGTATATCTCCTTCTTAAAGTTAA ACAAAATTATTTCTAGAGG-3’. The resulting PCR products were treated with DpnI enzyme and were purified by gel-extraction kit by the manufacturer’s instructions. In addition, distinct linear DNA Inserts encoding different proteins were amplified by PCR from the plasmids of pET26b-SDAB-Her2 nanobody, pGEX5-GST, pCMV6-AC-GFP, pCW-Endo-A, and pET26b-Endo-S2 plasmids respectively with the listed primers. Finally, the constructed linearized CPD-containing pET22b DNA and distinct DNA inserts were mixed respectively (the molar ratio of the DNA insert to linear vector was 2:1) in a seamless cloning master mix, followed by diluting to a specific volume using sterilized ddH2O. The reaction mixture was incubated at 50 °C for 20 min and placed on ice for 2 min immediately.
Forward primer of Her2 nanobody:
5’-GAAGGAGATATACATATGCAAGTTCAGCTGCAGGAAAGCGGT-3’
Reverse primer of Her2 nanobody:
5’-CGCTAATGCGTCGACACCACCGCTGCTCACGGTCACCTGGGT-3’
Forward primer of GST:
5’-GAAGGAGATATACATATGTCCCCTATACTAGGTTATTGGAAAATTAAGGG-C-3’
Reverse primer of GST:
5’-CGCTAATGCGTCGACTTTTGGAGGATGGTCGCCACCACCAAA-3’
Forward primer of GFP:
5’-GAAGGAGATATACATATGGAGAGCGACGAGAGCGGC-3’
Reverse primer of GFP:
5’-CGCTAATGCGTCGACTTCTTCACCGGCATCTGCATC-3’
Forward primer of Endo-A:
5’-GAAGGAGATATACATATGTCTACGTACAACGGCCCGCTG-3’
Reverse primer pf Endo-A:
5’-CGCTAATGCGTCGACAAACGAGCCGCTTTTTATGTCGATCAT-3’
Forward primer of Endo-S2:
5’-CACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAG-3’
Reverse primer pf Endo-S2:
5’- CCATCCGCTAATGCGTCGACAGCTTTAGCATCATCCATCT-3’
Forward primer of Her2 nanobody (N-flag tag):
5’-GATTACAAGGACGACGATGACAAGCAAGTTCAGCTGCAGGAAAGCGGTGGT-3’
Reverse primer of Her2 nanobody (N-flag tag):
5’-CTTGTCATCGTCGTCCTTGTAATCCATATGTATATCTCCTTCTTAAAGTTA-3’
Expression and purification of the CPD-tagged proteins
The above seamless cloning reaction mixtures were transformed into competent E. coli Trans5α respectively. All constructed plasmids were confirmed by DNA sequencing, and either the amino acids sequence or DNA sequence could be found in the Supplementary Information (section 4, protein sequences). The confirmed plasmids with DNA encoding CPD-tagged Endo-F3(D165A), Her2 nanobody, GST, GFP, Endo-A, or Endo-S2 was transformed into the E. coli BL21 (DE3) cells for overexpression respectively. The cells were first grown in 5 mL of LB medium containing 0.1 mg/mL ampicillin at 37 °C for 6 h. When the OD600 of 0.6 was reached, 100 μL of cells were transformed into 100 mL of LB medium containing 0.1 mg/mL ampicillin and the mixture was incubated for another 8 h at 37 °C until the OD600 reached 0.6–1.0, then 0.5 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the above cells to induce the expression of the target proteins. The induced cells were incubated at 20 °C for 12 h and harvested by centrifugation at 4 °C and 12000x g for 20 min. The cell lysates were collected via ultrasonication. Distinct CPD-tagged proteins were purified using a Ni-NTA agarose from the supernatant, and the concentration of each protein was measured using a Nanodrop instrument.
Preliminary evaluation of specific modification of CPD-tagged protein
Endo-F3(D165A)-CPD (100 μM), 3-adizopropylamine (50 mM or without), 100 μM InsP6 was incubated in PBS at 4 °C for 30 min. Or Endo-F3(D165A)-CPD (100 μM) and InsP6 (100 μM) was first incubated in PBS at 4 °C for 2 h, then additional 3-adizopropylamine (50 mM) was added to the reaction mixture which was further incubated at 4 °C for 30 min. All the reactions were monitored by ESI-TOF-MS.
General procedures for optimized C-terminal modification and purification
CPD-tagged proteins (50–100 μM), amines or hydrazine (5 mM), InsP6 (5 μM), and DMAP (5 mM) were mixed in NaOAc buffer (50 mM, final pH 8.0) at 4 °C for 3 h. The reaction was monitored by ESI-TOF-MS. After completion, the Ni-NTA agarose in PBS was added to remove free CPD with a His6-tag. After agitation for 2 h, the filtrate was collected and concentrated by centrifugal filtration tube (3 kDa or 10 kDa, Millipore). The buffer was exchanged to PBS to facilitate the removal of excess amine, DMAP and InsP6, and to give the C-terminal modification proteins. The ESI-TOF-MS profiles of the C-terminus-modified proteins are listed in Supplementary Table 1 in the supplementary information.
Modification of Her2 Nanobody with GlcNAc
A solution of Her2 Nb-NHNH2 (50 μM), GlcNAc (1 M) in NaOAc buffer (100 mM, pH 4.0) was incubated at 37 °C for 10 h. ESI-TOF-MS of Her2 Nb-GlcNAc: calcd. 13487.1; found 13487.0.
Procedures for native chemical ligation
A solution of Her2 Nb-NHNH2 (50 μM), NaNO2 (300 μM) in glycine (0.2 M, pH 3.0) was incubated on ice for 30 min. Then MPAA was added at a final concentration of 20 mM, and the pH was adjusted to 6.0–6.5. The reaction mixture was kept on ice for another 15 min. Then, the cysteine or peptide H-CDYKDDDDK-OH (50 mM) was added and the pH was adjusted to 7.0–7.5 by 500 mM NaOH, and the reaction mixture was incubated at 25 °C for another 2 h. ESI-TOF-MS of Her2 Nb-Cys: calcd. 13373.0; found 13373.0. ESI-TOF-MS of Her2 Nb-Flag: calcd. 14534.2; found 14534.0.
Preparation of the Her2 Nanobody-FITC conjugate
A solution of Her2 Nb-Az (100 μM), DBCO-FITC (500 μM) in PB buffer (50 mM, pH 7.4) was incubated at 37 °C for 2 h. The reaction was monitored by ESI-TOF-MS. The reaction mixture was purified by centrifugal filtration (10 kDa MWCO, Millipore). The buffer was exchanged to PBS to give the Her2 Nanobody-FITC conjugate in 1.0 mg/mL. ESI-TOF-MS of Her2 Nb-FITC: calcd. 14017.7; found 14017.0.
Preparation of the Her2 Nanobody-MMAE conjugate
A solution of Her2 Nb-azide (100 μM), BCN-Lys(PEG24)-VC-PAB-MMAE (500 μM) in PB buffer (50 mM, pH 7.4) was incubated at 37 °C overnight. The reaction was monitored by ESI-TOF-MS. The reaction mixture was purified by centrifugal filtration (10 kDa MWCO, Millipore). The buffer was exchanged to PBS to give the Her2 Nanobody-MMAE conjugate. ESI-TOF-MS of Her2 Nb-MMAE: calcd. 15923.1; found 15923.0.
Preparation of the bispecific antibody
A solution of DBCO-tagged Trastuzumab (5 mg/mL), Her2 Nb-azide (133 μM) in PB buffer (50 mM, pH 7.4) was incubated at 37 °C for 8 h. The progress of the reaction was monitored by SDS-PAGE. In addition, a solution of Mtz-tagged Trastuzumab (5 mg/mL), Her2 Nb-TCO (500 μM) in 50 mM PB buffer (pH 7.4) was incubated at 37 °C for 8 h. The reaction was monitored by SDS-PAGE.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
Methods and all relevant data are available in Supplementary Information. The source data underlying Figs. 4b, c, d, e, f, and i, and Supplementary Figs. S24 are provided as a Source Data file. Source data are provided with this paper.
References
Harris, J. M. & Chess, R. B. Effect of pegylation on pharmaceuticals. Nat. Rev. Drug Discov. 2, 214–221 (2003).
Reddi, R. N. et al. Site-Specific Labeling of Endogenous Proteins Using CoLDR Chemistry. J. Am. Chem. Soc. 143, 20095–20108 (2021).
Wolter, M. et al. Fragment-Based Stabilizers of Protein-Protein Interactions through Imine-Based Tethering. Angew. Chem. Int. Ed. 59, 21520–21524 (2020).
Hoyt, E. A., Cal, P. M. S. D., Oliveira, B. L. & Bernardes, G. J. L. Contemporary approaches to site-selective protein modification. Nat. Rev. Chem. 3, 147–171 (2019).
Fischer, N. H., Oliveira, M. T. & Diness, F. Chemical modification of proteins - challenges and trends at the start of the 2020s. Biomater. Sci.-Uk 11, 719–748 (2023).
Jiang, H. F., Chen, W. J., Wang, J. & Zhang, R. S. Selective N-terminal modification of peptides and proteins: Recent progresses and applications. Chin. Chem. Lett. 33, 80–88 (2022).
Morgan, H. E., Turnbull, W. B. & Webb, M. E. Challenges in the use of sortase and other peptide ligases for site-specific protein modification. Chem. Soc. Rev. 51, 4121–4145 (2022).
Boutureira, O. & Bernardes, G. J. L. Advances in Chemical Protein Modification. Chem. Rev. 115, 2174–2195 (2015).
Haque, M., Forte, N. & Baker, J. R. Site-selective lysine conjugation methods and applications towards antibody-drug conjugates. Chem. Commun. 57, 10689–10702 (2021).
Choi, H., Kim, M., Jang, J. & Hong, S. Visible-Light-Induced Cysteine-Specific Bioconjugation: Biocompatible Thiol-Ene Click Chemistry. Angew. Chem. Int. Ed. 59, 22514–22522 (2020).
Laserna, V. et al. Protein Conjugation by Electrophilic Alkynylation Using 5-(Alkynyl)dibenzothiophenium Triflates. Bioconjug. Chem. 32, 1570–1575 (2021).
Jia, S., He, D. & Chang, C. J. Bioinspired Thiophosphorodichloridate Reagents for Chemoselective Histidine Bioconjugation. J. Am. Chem. Soc. 141, 7294–7301 (2019).
Wan, C. et al. Histidine-specific bioconjugation via visible-light-promoted thioacetal activation. Chem. Sci. 13, 8289–8296 (2022).
Maruyama, K. et al. Protein Modification at Tyrosine with Iminoxyl Radicals. J. Am. Chem. Soc. 143, 19844–19855 (2021).
Zhang, Y., Park, K. Y., Suazo, K. F. & Distefano, M. D. Recent progress in enzymatic protein labelling techniques and their applications. Chem. Soc. Rev. 47, 9106–9136 (2018).
Antos, J. M. et al. Site-specific N- and C-terminal labeling of a single polypeptide using sortases of different specificity. J. Am. Chem. Soc. 131, 10800–10801 (2009).
Morgan, H. E., Arnott, Z. L. P., Kaminski, T. P., Turnbull, W. B. & Webb, M. E. Combined Application of Orthogonal Sortases and Depsipeptide Substrates for Dual Protein Labeling. Bioconjug. Chem. 33, 2341–2347 (2022).
Cong, M. et al. Direct N- or C-Terminal Protein Labeling Via a Sortase-Mediated Swapping Approach. Bioconjug. Chem. 32, 2397–2406 (2021).
Meyer, C., Liebscher, S. & Bordusa, F. Selective Coupling of Click Anchors to Proteins via Trypsiligase. Bioconjugate. Chem. 27, 47–53 (2016).
Schumacher, D. et al. Versatile and Efficient Site-Specific Protein Functionalization by Tubulin Tyrosine Ligase. Angew. Chem. Int. Ed. 54, 13787–13791 (2015).
Nguyen, G. K. et al. Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis. Nat. Chem. Biol. 10, 732–738 (2014).
Harris, K. S. et al. Efficient backbone cyclization of linear peptides by a recombinant asparaginyl endopeptidase. Nat. Commun. 6, 10199 (2015).
Yang, R. et al. Engineering a Catalytically Efficient Recombinant Protein Ligase. J. Am. Chem. Soc. 139, 5351–5358 (2017).
Jackson, M. A. et al. Molecular basis for the production of cyclic peptides by plant asparaginyl endopeptidases. Nat. Commun. 9, 2411 (2018).
Rehm, F. B. H. et al. Enzymatic C-Terminal Protein Engineering with Amines. J. Am. Chem. Soc. 143, 19498–19504 (2021).
Zhang, D. et al. Asparaginyl Endopeptidase-Mediated Protein C-Terminal Hydrazinolysis for the Synthesis of Bioconjugates. Bioconjug. Chem. 33, 238–247 (2022).
Zhu, T. et al. Creating an Unusual Glycine-Rich Motif in a Peptide Amidase Leads to Versatile Protein C-Terminal Traceless Functionalization. ACS Catal. 8019–8026 (2022).
Komiya, C. et al. Traceless synthesis of protein thioesters using enzyme-mediated hydrazinolysis and subsequent self-editing of the cysteinyl prolyl sequence. Chem. Commun. (Camb.) 55, 7029–7032 (2019).
Li, R. et al. Traceless enzymatic protein synthesis without ligation sites constraint. Natl Sci. Rev. 9, nwab158 (2022).
Bloom, S. et al. Decarboxylative alkylation for site-selective bioconjugation of native proteins via oxidation potentials. Nat. Chem. 10, 205–211 (2018).
Muir, T. W., Sondhi, D. & Cole, P. A. Expressed protein ligation: A general method for protein engineering. Proc. Nat. Acad. Sci. Usa. 95, 6705–6710 (1998).
Shah, N. H. & Muir, T. W. Inteins: Nature’s Gift to Protein Chemists. Chem. Sci. 5, 446–461 (2014).
Thompson, R. E. & Muir, T. W. Chemoenzymatic Semisynthesis of Proteins. Chem. Rev. 120, 3051–3126 (2020).
Thom, J., Anderson, D., McGregor, J. & Cotton, G. Recombinant protein hydrazides: application to site-specific protein PEGylation. Bioconjug. Chem. 22, 1017–1020 (2011).
Chiang, M. J. et al. An Fc domain protein-small molecule conjugate as an enhanced immunomodulator. J. Am. Chem. Soc. 136, 3370–3373 (2014).
Qiao, Y. et al. Expressed Protein Ligation without Intein. J. Am. Chem. Soc. 142, 7047–7054 (2020).
Lupardus, P. J., Shen, A., Bogyo, M. & Garcia, K. C. Small Molecule-Induced Allosteric Activation of the Vibrio cholerae RTX Cysteine Protease Domain. Science 322, 265–268 (2008).
Prochazkova, K. & Satchell, K. J. Structure-function analysis of inositol hexakisphosphate-induced autoprocessing of the Vibrio cholerae multifunctional autoprocessing RTX toxin. J. Biol. Chem. 283, 23656–23664 (2008).
Shen, A. et al. Mechanistic and structural insights into the proteolytic activation of Vibrio cholerae MARTX toxin. Nat. Chem. Biol. 5, 469–478 (2009).
Giddens, J. P., Lomino, J. V., Amin, M. N. & Wang, L. X. Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins. J. Biol. Chem. 291, 9356–9370 (2016).
Shen, A. et al. Simplified, enhanced protein purification using an inducible, autoprocessing enzyme tag. PLoS One 4, e8119 (2009).
Biancucci, M. et al. New ligation independent cloning vectors for expression of recombinant proteins with a self-cleaving CPD/6xHis-tag. BMC Biotechnol. 17, 1 (2017).
Zou, X. et al. Correction: Enhanced transglycosylation activity of an Endo-F3 mutant by ligand-directed localization. Org. Biomol. Chem. 20, 5638 (2022).
Zheng, J. S., Tang, S., Qi, Y. K., Wang, Z. P. & Liu, L. Chemical synthesis of proteins using peptide hydrazides as thioester surrogates. Nat. Protoc. 8, 2483–2495 (2013).
Zeng, Y. et al. A Traceless Site-Specific Conjugation on Native Antibodies Enables Efficient One-Step Payload Assembly. Angew. Chem. Int. Ed. 61, e202204132 (2022).
Pihl, R., Zheng, Q. & David, Y. Nature-inspired protein ligation and its applications. Nat. Rev. Chem. 7, 234–255 (2023).
Xia, Y. et al. A Cascade Enzymatic Reaction Scheme for Irreversible Transpeptidative Protein Ligation. J. Am. Chem. Soc. 145, 6838–6844 (2023).
Nguyen, G. K., Cao, Y., Wang, W., Liu, C. F. & Tam, J. P. Site-Specific N-Terminal Labeling of Peptides and Proteins using Butelase 1 and Thiodepsipeptide. Angew. Chem. Int. Ed. 54, 15694–15698 (2015).
Cao, Y., Nguyen, G. K., Chuah, S., Tam, J. P. & Liu, C. F. Butelase-Mediated Ligation as an Efficient Bioconjugation Method for the Synthesis of Peptide Dendrimers. Bioconjug. Chem. 27, 2592–2596 (2016).
Rehm, F. B. H. et al. Site-Specific Sequential Protein Labeling Catalyzed by a Single Recombinant Ligase. J. Am. Chem. Soc. 141, 17388–17393 (2019).
Tang, T. M. S. et al. Use of an asparaginyl endopeptidase for chemo-enzymatic peptide and protein labeling. Chem. Sci. 11, 5881–5888 (2020).
Rehm, F. B. H., Tyler, T. J., Yap, K., Durek, T. & Craik, D. J. Improved Asparaginyl-Ligase-Catalyzed Transpeptidation via Selective Nucleophile Quenching. Angew. Chem. Int. Ed. 60, 4004–4008 (2021).
Acknowledgements
This work was supported by the National Key Research and Development Plan grant (2021YEE0200500), the Natural Science Foundation of China (NSFC, No. 92153301, 82003574, 82204183, 22277126, and 82325045), the Shanghai Sail Program (No. 22YF1457400), the Special Research Assistant Program (Chinese Academy of Sciences, CAS), the Hangzhou innovation and entrepreneurship leading team project (No. TD2020005), the China Postdoctoral Science Foundation (No. 2021M700158), and the Lingang Laboratory (No. LG-QS-202206-03). F. Tang gratefully acknowledge the support of the SANOFI Scholarship Program.
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Y.Z. performed the identification and optimization of CPD-mediated C-terminal azidation, tested different amine substrates and proteins, and constructed the biparatopic antibody. W.S. constructed the fusion proteins of EndoS2-CPD, Endo-A-CPD, GST-CPD, Her2 Nb-CPD, and GFP-CPD, prepared the nanobody-drug conjugates and determined the in vitro activity. Z.L. prepared the nanobody-fluorescence conjugate and performed the confocal microscopy. Y. W., L.L., J. H., and M.L. gave assistance in expressing POI-CPDs. H.X., and W.Z., helped in the synthesis of chemical substrates and condition optimization. W.H. and F.T. conceived the idea, designed the experiments, supervised the project, analyzed the data, and wrote the manuscript. Y.Z. and W.S. contributed equally to this work.
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Zeng, Y., Shi, W., Liu, Z. et al. C-terminal modification and functionalization of proteins via a self-cleavage tag triggered by a small molecule. Nat Commun 14, 7169 (2023). https://doi.org/10.1038/s41467-023-42977-x
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DOI: https://doi.org/10.1038/s41467-023-42977-x