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. 2026 Feb 9;6(2):943–954. doi: 10.1021/jacsau.5c01395

“Lys–Leu” Motif Empowers Expedient OaAEP1-Catalyzed Generation of N‑Terminal Cysteine Recombinant Proteins for Bioconjugation and Semisynthesis

Tingting Cui †, Na Liu †, Jian Sun †, Junjiang Li †, Guo-Chao Chu †,*, Yi-Ming Li †,‡,*
PMCID: PMC12933375  PMID: 41755832

Abstract

Site-specific protein modification enables precise functionalization for applications ranging from live-cell imaging to therapeutic development. Although N-terminal cysteine (N-Cys)-based modification exhibits excellent specificity, naturally occurring N-Cys is rare, and existing methods are usually limited by factors such as narrow substrate scopes, incompatibility with inclusion bodies, or residual exogenous sequences. Here, we report a highly efficient and versatile method for generating free N-Cys proteins through “Lys–Leu” (K–L) motif-assisted OaAEP1-catalyzed cleavage. This approach enhances cleavage efficiency across all 20 natural amino acids at the P2′ position, accommodates diverse substrates (including challenging inclusion body proteins), and enables streamlined one-pot in situ generation and functionalization, as exemplified by fluorescein/biotin labeling and dual modifications. Furthermore, we successfully applied this strategy to the traceless semisynthesis of histone H3Q5ser (serotonylated at Gln5). Our work establishes a robust platform for site-specific protein engineering and expands the functional versatility of OaAEP1 in synthetic biology and chemical biology applications.

Keywords: site-specific protein modification, N-terminal cysteine protein, OaAEP1-catalyzed cleavage, “Lys–Leu” motif, chemical protein synthesis

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Introduction

Protein-selective bioconjugation technology enables precise functionalization of proteins by introducing functional groups at defined protein sites. − For example, site-specific fluorescent labeling of proteins facilitates live-cell imaging; , targeted installation of post-translational modifications (PTMs) aids in dissecting how PTMs regulate protein function; − and site-specific conjugation supports the development of antibody-drug conjugates for therapeutics. Among available strategies, bioorthogonal modification through selective reactions between electrophilic reagents, such as aldehydes, thioesters, , and 2-cyanobenzothiazole (CBT), − and protein N-terminal cysteine (N-Cys) is particularly effective. − This is attributed to the protein N-terminus being typically solvent-exposed, such that modifications at this site minimally perturb protein folding and function. However, natural N-Cys residues are extremely rare in proteins, necessitating protein engineering strategies to generate N-Cys-containing proteins.

Although the direct preparation of N-Cys proteins using the Escherichia coli expression system is operationally simple, the resulting N-Cys proteins tend to react with endogenous pyruvate to form thiazolidine derivatives, which interferes with subsequent modifications. , Currently, the most widely used method involves introducing a protease-selective recognition motif at the N-terminus of proteins, followed by protease cleavage to retain N-Cys. Among enzyme-catalyzed methods for preparing N-Cys proteins (e.g., using factor Xa, TEV protease, SUMO protease, etc.), the cleavage efficiency varies significantly depending on the structure of the target protein, and most are incompatible with inclusion body proteins. , A recent advancement involves fusing an MPCGHKP-based sequence to the N-terminus of the target protein (sometimes with an additional flanking GSSGSS linker), followed by sequential treatment with methionine aminopeptidase and proline aminopeptidase (ProAP). Although efficient and versatile, this method leaves a long peptide “recognition motif” (GHKP or GHKPGSSGSS, at least 4 amino acids) adjacent to N-Cys (Scheme a). Further reducing exogenous amino acid incorporation would minimize the interference of modifications on protein structure and function and even achieve traceless proteins semisynthesis.

1. Methods for Preparing N-Cys Proteins by Protease Cleavage .

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a (a) The strategy for preparing N-Cys proteins via ProAP. (b) “Lys–Leu” motif-promoted OaAEP1-catalyzed strategy for preparing N-Cys proteins.

Herein, we report a novel and efficient OaAEP1-catalyzed method for efficiently preparing N-terminal cysteine proteins, which requires only one additional amino acid at the target N-terminus (Scheme b). The key is the introduction of a “Lys–Leu” (K–L) motif at the N-terminus adjacent to the OaAEP1 recognition motif Asn-Cys-P2′ (P1↓P1′–P2′). This modification significantly enhances the cleavage efficiency of OaAEP1 and broadens its substrate scope, enabling it to act on all 20 natural amino acids at the P2′ position. Using this method, various N-terminal cysteine proteins with fewer introduced exogenous sequences can easily be obtained, enabling one-pot site-specific labeling with fluorescein and biotin and even dual functionalization. Furthermore, this method is applicable to inclusion body proteins, leading to the successful traceless chemical semisynthesis of H3Q5ser.

Materials and Methods

Plasmid Construction

The complementary DNA of OaAEP1, H6-(GGS)2-KLNCL-Ub, H6-(GGS)2-KLNCL-MBP, and H6(GGS)2KLN-H3 (34–135) was synthesized by GenScript Biotech (Nanjing, China). Their sequences were optimized to suit overexpression in E. coli.

Protein Expression and Purification

The expression and purification methods of OaAEP1 were consistent with previously described methods. Detailed procedures are provided in the Supporting Information. The results of SDS-PAGE analysis for activated OaAEP1 are shown in Figure S3. Plasmids carrying the H6-(GGS)2-KLNCL-Ub gene (vector pET-22b­(+), ampicillin resistance) and H6-(GGS)2-KLNCL-MBP gene (vector pMAL-c4x, kanamycin resistance) were separately transformed into E. coli BL21 (DE3) competent cells, while plasmids containing the H6(GGS)2KLN-H3 (34–135) gene (vector pET-15b) were also transformed into the same competent cells; for each transformation, a single colony was inoculated into 10 mL of LB medium supplemented with the corresponding antibiotic (10 μg/mL ampicillin for H6-(GGS)2-KLNCL-Ub and H6(GGS)2KLN-H3 (34–135), 10 μg/mL kanamycin for H6-(GGS)2-KLNCL-MBP) and cultured overnight at 37 °C. The overnight cultures were then transferred to 1 L of fresh LB medium containing the respective antibiotics at the same concentrations and incubated at 37 °C until the OD600 reached 0.6–0.8, at which point protein expression was induced by adding 0.5 mM isopropyl β-d-thiogalactopyranoside (IPTG): H6-(GGS)2-KLNCL-Ub and H6(GGS)2KLN-H3 (34–135) expression was continued at 37 °C for 14–16 h, while H6-(GGS)2-KLNCL-MBP expression was incubated at 16 °C for another 14–16 h. After induction, cells were harvested by centrifugation, with those expressing H6-(GGS)2-KLNCL-Ub and H6-(GGS)2-KLNCL-MBP resuspended in resuspension buffer (25 mM HEPES, 150 mM NaCl, pH 7.4) and those expressing H6(GGS)2KLN-H3 (34–135) resuspended in lysis buffer (50 mM Tris, 300 mM NaCl, 1 mM EDTA, pH 8.0), followed by lysis via ultrasonication on ice for all samples; the cell lysates were centrifuged at 4 °C for 30 min to collect supernatants, with the supernatant from H6(GGS)2KLN-H3 (34–135) cultures processed further by dissolving the resulting pellet in unfolding buffer (6 M guanidine hydrochloride, 20 mM Tris, pH 7.5), sonicating for 4 h, stirring overnight at room temperature, and centrifuging at high speed the next day to collect an additional supernatant. All supernatants were purified via nickel-affinity chromatography, with the H6(GGS)2KLN-H3 (34–135) sample using three specific buffers (25 mM Tris with 150 mM NaCl, pH 7.4; 6 M urea with 25 mM Tris, 150 mM NaCl, 20 mM imidazole, pH 7.4; and 6 M urea with 25 mM Tris, 150 mM NaCl, 250 mM imidazole, pH 7.4); purified H6-(GGS)2-KLNCL-Ub and H6-(GGS)2-KLNCL-MBP proteins were dialyzed against dialysis buffer (25 mM HEPES, 150 mM NaCl, pH 7.4) for 12 h, then concentrated and stored at −80 °C, while the purified H6(GGS)2KLN-H3 (34–135) sample was separated by HPLC and lyophilized.

Peptide Synthesis Methods

All peptides utilized in this study were synthesized via standard Fmoc-SPPS (Fmoc solid-phase peptide synthesis) on Rink amide resin using a Liberty Blue 2.0 automated microwave peptide synthesizer (CEM Corporation, North Carolina, USA).

OaAEP1-Catalyzed Generation of N-Cys Peptides

The model peptide NH2-MKLNCXPYRR-CONH2 (X = 20 natural amino acids) at a concentration of 1 mM was dissolved in PBS solution containing 1 mM TCEP. Hydrazine hydrate (200 mM) and OaAEP1 (5 μM) were added, and the mixture was incubated at pH 7.0 and 25 °C. For peptides with X = Leu, Ile, Met, Val, Phe, or Cys, the reaction was allowed to proceed for 30 min; for the remaining model peptides, the reaction duration was 2.5 h. The reaction was terminated by the addition of 1‰ trifluoroacetic acid. For the one-pot N-terminal modification of proteins, H6-(GGS)2-KLNCL-Ub/H6-(GGS)2-KLNCL-MBP was dissolved in PBS buffer containing 1 mM TCEP to a final concentration of 50 μM. Subsequently, 100 μM Biotin-CBT/FITC-CBT and 0.25 μM OaAEP1 were added, and the reaction was conducted at pH 7 and 25 °C. For the construction of dual-modified proteins, H6-(GGS)2-KLNCL-Ub was dissolved in PBS buffer containing 1 mM TCEP to a final concentration of 300 μM. After adding 200 mM hydrazine hydrate and 0.6 μM OaAEP1, the reaction was carried out at pH 7 and 25 °C for 2 h. Subsequently, the reaction solution was purified using reverse-binding Ni-NTA to obtain CL-Ub, which was concentrated and used for NCL. For H6(GGS)2KLN-H3 (34–135), the protein was dissolved in PBS buffer containing 1 mM TCEP to a final concentration of 50 μM. After adding 200 mM hydrazine hydrate and 2 μM OaAEP1, the reaction was conducted at pH 7.0 and 25 °C for 5 h. Subsequently, the reaction solution of H6(GGS)2KLN-H3 (34–135) was purified using a reverse-binding Ni-NTA column to obtain H3 (34–135), which was concentrated and used for NCL.

Cell Imaging

HeLa cells were seeded into sterile 35 mm glass-bottom culture dishes and cultured at 37 °C in a humidified atmosphere containing 5% carbon dioxide for 24 h to ensure proper cell attachment. The culture medium consisted of DMEM supplemented with 10% FBS, 100 units/mL of penicillin, and 0.1 mg/mL of streptomycin. After the attachment period, the cells were incubated with DMEM containing FITC-Ub-cR10 for 2 h under the same culture conditions. Following incubation, the cells were thoroughly washed three times with PBS. Fluorescence imaging was performed using a Zeiss LSM 880 AxioObserver confocal laser scanning microscope equipped with ZEN software. Cell nuclei were detected via Hoechst 33258 fluorescence using a 405 nm ultraviolet laser, while FITC-Ub-cR10 was detected by using a 543 nm green laser. A Plan Apochromat 60×/1.4 Oil DIC M27 objective was used for imaging. Laser power and pixel resolution settings were carefully adjusted to minimize photobleaching and ensure optimal image quality.

Mass Spectrometry Analysis of H3Q5ser Modification Sites

Target gel bands were cut out from the gel, after which in-gel digestion was performed at 37 °C overnight. Next, the peptide was extracted twice using a buffer composed of 0.1% TFA and 50% aqueous acetonitrile and then dried with a SpeedVac (Thermo Scientific). Peptide was subsequently redissolved in 0.1% aqueous TFA and analyzed by using a Thermo Orbitrap Fusion mass spectrometer.

Results and Discussion

The “K–L” Motif Promotes OaAEP1-Catalyzed Generation of N-Terminal Cysteine Peptides

Our study began with the preparation of N-Cys peptides using the [Cys247Ala]OaAEP1 mutant (hereafter referred to as OaAEP1), , an enzyme that recognizes the tripeptide motif Asn-Cys-P2′ (P1↓P1′–P2′) ,, and exhibits higher catalytic activity than the wild-type. We synthesized a series of model peptides NH2-MNCXPYRR-CONH2 (P2′ = X = Leu, Ile, Met, Figure S4) that conform to OaAEP1 substrate preference. , These peptides were dissolved in PBS buffer (pH 7.0) at a final concentration of 1 mM, and the reaction was conducted at 25 °C with 200 mM hydrazine hydrate (as a reaction promoter) and 5 μM OaAEP1. HPLC analysis showed that all peptides were converted to N-Cys target products (NH2-CXPYRR-CONH2, X = Leu, Ile, Met) within 30 min, with conversion rates exceeding 70%. Subsequently, we tested the efficiency of preparing N-Cys peptides when P2′ was a nonpreferred amino acid for OaAEP1 (e.g., Val 1a, Cys 1b, Ala 1c, His 1d) under the same conditions (Figure a) and observed that the conversion rates of N-Cys peptides (2a–2d) were all below 40% (Figure b). This low yield limits the versatility of OaAEP1 in the preparation of N-Cys peptides and proteins.

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Discovery and characterization of the “K–L” motif promoting OaAEP1-catalyzed generation of N-Cys peptides. (a) OaAEP1-catalyzed generation of N-Cys peptides from 1a–1d. Reaction for 0.5 or 2.5 h. (b) Analytical HPLC of the process for OaAEP1-catalyzed generation of N-Cys peptides from 1a–1d. (c) The “K–L” motif promoting OaAEP1-mediated generation of N-Cys peptides from 4a–4d. Reaction for 0.5 or 2.5 h. (d) Analytical HPLC traces of the process for the “K–L” motif promoting OaAEP1-mediated generation of N-Cys peptides from 4a–4d. (e) Comparison of conversion rates of OaAEP1-mediated generation of N-Cys peptides from model peptides with and without the “K–L” motif. (f) Comparison of conversion rates of OaAEP1-mediated generation of 2b from 1b and 4b within 30 min. “*” indicates hydrolysis of 5.

Recently, Craik et al. found that the presence of Leu adjacent to a Lys residue in peptides/proteins significantly enhances OaAEP1 recognition of Lys and promotes isopeptide bond formation. , Building on this insight, we synthesized a second series of model peptides (NH2-MKLNCXPYRR-CONH2, X = various amino acids) by introducing a K–L motif at the N-terminus adjacent to the Asn-Cys-P2′ sequence (Figure c). HPLC analysis of OaAEP1-catalyzed cleavage of these peptides under the same reaction conditions showed that when P2′ was a nonpreferred amino acid (e.g., Val 4a, Cys 4b, or Ala 4c), the conversion rate of N-Cys peptides significantly increased from <40% to >90%; when P2′ was a preferred amino acid (e.g., Leu, Ile, or Met (Figure d,e)), the conversion rate further reached ∼100%. Further studies showed that within the same reaction time (30 min), the conversion rate of model peptides containing the “K–L” motif exceeded 95%, significantly higher than that of peptides lacking the motif (<40%), indicating a more efficient conversion rate (Figure f).

Exploring the Role of the “K–L” Motif in OaAEP1-Mediated Substrate Recognition

To investigate how the “K–L” motif facilitates substrate recognition by OaAEP1, we first synthesized the self-quenched fluorescent peptides Abz-AEEA-KLNCIG-Y­(3NO2)­RR and Abz-AEEA-NCIG-Y­(3NO2)­RR and performed enzyme kinetic assays to determine the key kinetic parameters (K M, k cat, and k cat/K M ratio) , (Figure S9). Our results revealed that the peptide lacking the K–L motif had a K M value of 72.38 ± 9.61 μM, a k cat value of 0.044 ± 0.19 s–1, and a k cat/K M ratio of 608 M–1 s–1. In contrast, introduction of the K–L motif results in a significant decrease in K M to 25.75 ± 2.56 μM, an increase in k cat to 0.14 ± 0.28 s–1, and an elevation in the k cat/K M ratio to 5437 M–1 s–1 (Figure a).

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Exploration of the “K–L” motif’s role in OaAEP1-mediated substrate recognition. (a) Kinetic parameters for OaAEP1-mediated cleavage of Abz-AEEA-KLNCIG-Y­(3NO2)­RR and Abz-AEEA-NCIG-Y­(3NO2)­RR. (n = 3; SEM, standard error of the mean). (b) SPR analysis of the binding interactions between OaAEP1 and two peptide substrates: one containing the K–L motif (4a) and the other lacking the K–L motif (1a). (c) The structural model depicts the complex of OaAEP1 with 4a. OaAEP1 is presented in cartoon mode. The peptide ligand is shown in stick model: carbon atoms (yellow), nitrogen atoms (blue), oxygen atoms (red).

To further verify the effect of the K–L motif on the binding between the substrate and OaAEP1, we employed surface plasmon resonance (SPR) assays to measure the dissociation constant (K D) between OaAEP1 with the peptide substrate containing the K–L motif (4a) and that lacking the K–L motif (1a), which were 5 and 45.28 μmol/L, respectively (Figure b). These indicated that the K–L motif enhanced the binding affinity between the substrate and OaAEP1 by approximately 9-fold. Collectively, these results demonstrate that the K–L motif enhances substrate recognition by increasing the binding affinity between OaAEP1 and its substrate and improves catalytic efficiency by accelerating the enzymatic turnover rate.

Meanwhile, we performed molecular dynamics (MD) simulations to elucidate how the K–L motif alters the substrate recognition and catalytic efficiency. The MD results showed that the average binding free energy (ΔG bind) of the substrate containing the K–L motif (4a, Figure c) was −53.63 (kcal/mol Figure S11), which was lower than that of the substrate lacking the K–L motif (1a, ΔG bind = −48.87 kcal/mol, Figure S13). This indicates that the incorporation of the K–L motif enhances the stability of the OaAEP1-substrate complex, which could explain the improved substrate binding affinity and catalytic efficiency observed in our enzyme kinetic and SPR assays.

“K–L” Motif-Empowered OaAEP1 to Recognize All 20 Natural Amino Acids at the P2′ Position

After confirming that the “K–L” motif promotes OaAEP1-catalyzed generation of N-Cys peptides, we synthesized model peptides (NH2-MKLNCXPYRR-CONH2, X = 20 natural amino acids, 4a–4t) covering all 20 natural amino acids to test the versatility of this method under standard reaction conditions (1 mM peptide substrate, 200 mM hydrazine hydrate, 5 μM OaAEP1, PBS buffer containing 1 mM TCEP, pH 7.0, 25 °C) (Figure ). The results showed that for hydrophobic aliphatic amino acids (Leu, Met, Ile, and Phe), nearly quantitative conversion was achieved within 30 min (yield ∼100%); for nonpreferred neutral amino acids (Gly, Gln, and Thr), the conversion efficiency exceeded 70%, with Thr reaching over 90%; notably, conversion products could still be isolated even for charged amino acids (Arg, Glu, Asp, and Lys) and conformationally constrained proline, which are usually poorly recognized by OaAEP1.

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“K–L” motif-promoted OaAEP1-catalyzed strategy for preparing N-Cys peptides with different P2′ amino acids. Reaction condition: 1 mM peptides, 200 mM hydrazine hydrate, 5 μM OaAEP1, pH 7.0, 25 °C, reaction for 0.5 or 2.5 h.

We also found that in a typical OaAEP1-catalyzed hydrolysis reaction, the Tyr and Val residues at the P2′ position of the substrate exhibit poor tolerance (with a yield of less than 35%), whereas the cleavage efficiency can be increased to over 80% after the incorporation of the “K–L” motif. Meanwhile, for peptides containing Asp, Pro, Arg, or Glu at the P2′ position, even extending the reaction time to 8 h did not significantly enhance the conversion efficiency (Figure S8). These results demonstrate that introducing the “K–L” motif enables OaAEP1 to efficiently recognize all 20 natural amino acids at the P2′ position and generate N-cysteine peptides while significantly enhancing the enzyme’s catalytic efficiency and substrate compatibility.

To investigate more combinations of residues at the P2 and P3 positions, we synthesized a series of model peptide substrate substitutes for the K and L residues individually and quantitatively analyzed the efficiency of their hydrolysis by OaAEP1 via chromatography (Figure S6). The results showed that although other residue combinations exerted a certain promoting effect on catalytic efficiency, the “K–L” motif exhibited the most significant enhancement effect.

One-Pot N-Terminal Modification of Proteins Using the “K–L”/OaAEP1-Catalyzed Strategy

Next, we applied the “K–L” motif-promoted, OaAEP1-mediated N-Cys generation strategy (K–L/OaAEP1-catalyzed strategy) to protein N-terminal modification (Figure a). Taking ubiquitin (Ub, ∼8 kDa) as an example, we first introduced an H6-(GGS)2-KLNCL fusion tag at its N-terminus (H6-(GGS)2-KLNCL-Ub, 6). The condensation reaction between 2-cyanobenzothiazole (CBT) and N-Cys is characterized by a fast reaction rate, mild conditions (neutral pH), and the efficient introduction of functional groups through CBT, making it a valuable method for site-specific protein modification. Based on this, we synthesized two functional derivatives, Biotin-CBT and FITC-CBT, aiming to achieve one-pot preparation and modification of N-Cys proteins by in situ capturing of N-Cys proteins released by enzymatic reactions. Purified 6 was dissolved in PBS buffer (pH 7.0) and reacted with OaAEP1 and Biotin-CBT at 25 °C for 5 h (Figure b). HPLC analysis showed complete disappearance of the 6 peak and emergence of a new characteristic peak (retention time = 21.2 min). ESI-MS identification confirmed that the molecular weight of this main peak matched the theoretical value of Biotin-modified Ub (7, HPLC yield >90%, Figure c). Using the same strategy, replacing Biotin-CBT with fluorescein-labeled CBT derivative (FITC-CBT) efficiently yielded FITC-labeled Ub (8), with product correctness verified by SDS-PAGE combined with fluorescent gel imaging and high-resolution mass spectrometry (HRMS) (Figure d–f).

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Performing one-pot N-terminal modification of proteins via the K–L/OaAEP1-catalyzed strategy. (a) Schematic diagram for performing one-pot N-terminal modification of proteins via the K–L/OaAEP1-catalyzed strategy. (b) Analytical HPLC traces of 7 generated by one-pot N-terminal biotin modification of Ub via a new strategy. (c) Structure and HRMS analysis of 7. (d) Analytical HPLC traces of 8 generated by one-pot N-terminal FITC modification of Ub. (e) Structure and HRMS analysis of 8. (f) SDS-PAGE analysis confirms the generation of 8 by one-pot N-terminal modification of proteins using a new strategy. (g) Analytical HPLC traces of 10 generated by one-pot N-terminal biotin modification of MBP. (h) Structure and HRMS analysis of 10. (i) Analytical HPLC traces of 11 generated by one-pot N-terminal FITC modification of MBP. (j) Structure and HRMS analysis of 11. (k) SDS-PAGE analysis confirms the generation of 11 by one-pot N-terminal modification of proteins.

To further evaluate the applicability of this one-step modification reaction to proteins with a high molecular weight (MW > 40 kDa), we construct the H6-(GGS)2-KLNCL-MBP (9) fusion protein (MBP, 45 kDa). 9 was incubated with OaAEP1 and Biotin-CBT/FITC-CBT at pH 7.0 and 25 °C. The modified products were characterized by analytical HPLC, SDS-PAGE combined with fluorescence imaging, and mass spectrometry, confirming the successful preparation of biotinylated (10) and FITC-labeled MBP (11) derivatives (HPLC yield >95%, Figure g–k). We also tested the cleavage efficiency toward protein MKLNCL-UbcH7 (20 kDa), which harbors an α-helical structure with large steric hindrance at its N-terminus. After incubating with OaAEP1 for 3 h, we successfully obtained the target product CL-UbcH7 using chromatographic separation, with a relative high yield (Figure S14). Collectively, these results demonstrate that the K–L/OaAEP1 strategy enables the efficient one-pot in situ generation and functionalization of diverse N-Cys proteins.

Application of a New Method to Dual Modification of Proteins

We further explored the application of this method for protein dual functionalization, such as constructing a cell-penetrating fluorescently labeled Ub for living cell imaging. First, 6 was cleaved with 5 μM OaAEP1 at 25 °C and pH 7.0, with the conversion rate nearly reached 100% after 2 h of reaction, and N-Cys-Ub (12) was obtained by nickel column purification (Figure a). Subsequently, 12 was first conjugated with synthesized FITC-AEEA-AEEA-CONHNH2 via hydrazides-based native chemical ligation (NCL) − to yield FITC-Ub (13), then covalently linked to 5,5′-dithiobis­(2-nitrobenzoic acid) (DTNB)-activated cell-penetrating peptide cR10 (TNB-cR10, 14) via disulfide bond formation, finally yielding the dual-functional protein FITC-Ub-cR10 (15) with cell permeability and fluorescent modification. The purity and correct molecular weight were confirmed by RP-HPLC and ESI-MS analyses (Figure b–d).

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Application of the K–L/OaAEP1-catalyzed strategy to dual modification of protein. (a) Schematic diagram of the generation of 13 via the K–L/OaAEP1-catalyzed strategy. (b) Schematic diagram of obtaining 15 via thiol–disulfide bond exchange. (c) HPLC and the ESI-MS analysis of 13. (d) HPLC and the ESI-MS analysis of 15. (e) Live-cell fluorescence imaging of HeLa cells treated with 20 μM 15 (visualized via FITC fluorescence in the green channel) with nuclei stained with Hoechst (blue channel), scale bar 20 μm. (f) MTT assay for cell viability analysis following treatment with 20 μM 15 for 1 h, with PBS treatment serving as a control. Peaks in ESI-MS marked with “#” corresponded to TFA adducts.

To validate cell permeability and biocompatibility, 20 μM 15 was incubated with HeLa cells in serum-free medium (37 °C, 5% CO2) for 1 h. After PBS washing, Hoechst 33342 (nuclear stain) was added for nuclear staining. Confocal laser scanning microscopy (CLSM) showed that in cells treated with 15, green fluorescence was evenly distributed in the cytoplasm and nucleus (Figure e). Meanwhile, Hoechst 33342 staining showed normal nuclear morphology, indicating that 20 μM 15 did not cause significant cytotoxicity during the cell uptake (Figure e). MTT assay further confirmed that 15 at this concentration had no significant effect on cell proliferation (Figure f).

K–L/OaAEP1-Catalyzed Strategy-Mediated Traceless Protein Semisynthesis

Enzyme-catalyzed protein semisynthesis offers advantages such as high efficiency and good biocompatibility but typically requires relatively large recognition motifs (containing multiple amino acids). Leveraging the K–L/OaAEP1 strategy’s flexibility for recognition sequences, we extended it to protein semisynthesisfocusing on histone H3Q5ser (serotonylated at Gln5), a newly discovered PTM involved in gene transcription regulation with no reported chemical synthesis to date. ,

To construct histone H3Q5ser, we divided it into two fragments, H3 (1–33) and H3 (34–135), and introduced a cysteine mutation at the Gly34 site (G34C) as the ligation site for NCL (Figure a,b). The first fragment H3 (1–33) was prepared by Fmoc solid-phase synthesis and then converted to a thioester via hydrazide. , The N-terminus of the second fragment, H3 (34–135), was fused with an H6(GGS)2KLN tag, where Asn and H3 Cys34-Val35 together form the OaAEP1-specific recognition motif (Asn–Cys–Val). We prepared the H6(GGS)2KLN-H3 34–135 (16) fusion protein using the E. coli expression system, dissolved the fusion protein expressed in inclusion bodies at a final concentration of 50 μM in PBS buffer (pH 7.0), added 200 mM hydrazine hydrate and 2 μM OaAEP1, and reacted at 25 °C for 5 h. HPLC and ESI-MS analyses showed that OaAEP1 specifically cleaved the Asn–Cys bond under these conditions, with a conversion rate of H3 34–135 (17) exceeding 80%. Subsequently, the H6(GGS)2KLN tag was removed by nickel column affinity chromatography, yielding the N-terminal cysteine-containing 17 with purity >95%, as confirmed by mass spectrometry (Figure c).

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K–L/OaAEP1 strategy-mediated traceless protein semisynthesis of H3Q5ser. (a) Amino acid sequence and serotonylation structure of H3Q5ser. (b) Schematic diagram of the generation of 18. (c) Analytical HPLC traces and ESI-MS analysis of 17 generated via the K–L/OaAEP1 strategy. (d) Analytical HPLC traces, ESI-MS, and SDS-PAGE analysis of 18 generated via NCL. (e) MS/MS spectra of 18. (f) SDS-PAGE analysis of purified H3Q5ser-containing histone octamers stained with Coomassie brilliant blue. (g) 4.5% Native gels analysis of the reconstituted H3Q5ser NCP stained with SYBR Gold.

For semisynthesis, the H3Q5ser (1–33) peptide hydrazide fragment was dissolved in 6 M Gn·HCl/0.2 M Na2HPO4 buffer (pH 2.3), activated by treatment with acetylacetone (5 equiv) and MPAA (25 equiv) at pH 1–2 for 1 h, and subsequently, 17 was added with the pH adjusted to 6.8 to initiate NCL. After reacting at room temperature for 5 h, HPLC and ESI-MS analyses confirmed successful synthesis of full-length H3Q5ser (18) without additional amino acids (Figure d). MS/MS analysis clearly showed that the serotonylation modification was specifically located at the glutamine residue at position 5 (Figure e).

To validate the biological functionality, we further constructed a nucleosome core particle (NCP) containing H3Q5ser modification. To this end, 18 was in vitro assembled with the other core histones into an octamer complex, which was purified by size-exclusion chromatography. SDS-PAGE analysis showed >95% purity and intact components (Figure f). The octamer was then reconstituted in vitro with the 147 bp 601 DNA sequence, and the resulting NCP was confirmed by native PAGE analysis (Figure g). Our results showed that H3Q5ser with serotonylation modification successfully participates in nucleosome assembly, yielding structurally intact NCP. To further verify the utility of this method for preparing native-sequence N-Cys proteins, we successfully generated the H2B (C18–125) fragment without any extraneous amino acid residues via OaAEP1 cleavage of His6(GGS)2KLN-H2B (C18–125), achieving an HPLC yield of 84% (Figure S14).

These results demonstrate that the K–L-assisted OaAEP1 enzyme-catalyzed N-Cys protein generation strategy, when applied to protein semisynthesis, requires at most 1 additional amino acid in the target protein (even enabling traceless semisynthesis) and can be directly applicable to unfolded proteins expressed in inclusion bodies. This avoids the renaturation step required in SUMO protease cleavage technology and issues such as incomplete cleavage due to incomplete renaturation, providing an important technical platform for chemical protein synthesis. −

Conclusions

In summary, we have developed a highly efficient and versatile method for N-terminal cysteine protein generation through “K–L” motif-assisted OaAEP1-catalyzed cleavage. This strategy addresses key limitations of existing approaches by enhancing enzymatic cleavage efficiency across all 20 natural amino acids at the P2′ position, enabling compatibility with diverse substrates (including challenging inclusion body proteins), and minimizing exogenous sequence residues to as few as one amino acid, thereby enabling near-traceless semisynthesis.

The utility of this method is demonstrated through streamlined one-pot in situ generation and functionalization of the N-Cys proteins. This is exemplified by site-specific labeling with fluorescein and biotin and the construction of dual-modified proteins with cell-penetrating and fluorescent properties. Notably, this strategy is compatible with the cleavage of inclusion body-expressed proteins while providing an opportunity to prepare N-terminal cysteine proteins free of any additional amino acid residues. Taking advantage of this feature, we have successfully applied this strategy to the traceless semisynthesis of histone H3Q5ser, thereby highlighting its potential in the preparation of complex protein variants.

By expanding the substrate scope and efficiency of OaAEP1-mediated cleavage, this work establishes a robust platform for site-specific protein engineering. It not only facilitates precise bioconjugation and semisynthesis but also broadens the functional versatility of OaAEP1. Moreover, it provides important insights into enzyme engineering by showing how strategic incorporation of the (K–L) motif can expand the substrate scope of OaAEP1. Although molecular dynamics have preliminarily revealed the mechanism underlying the enhanced reaction efficiency of the “K–L” motif, we plan to further elucidate the molecular mechanism in the future by solving the crystal structure of the OaAEP1-substrate complex during hydrolysis.

Supplementary Material

au5c01395_si_001.pdf (1.9MB, pdf)

Acknowledgments

This work was supported by NSFC (Nos. 22277020, 22227810 for Y.-M.L., 22407121 for G.-C.C.), Anhui Provincial Natural Science Foundation (No. 2508085JX004 for Y.-M.L., 2408085QB057 for G.-C.C.), Beijing Life Science Academy (BLSA, No: 2023000CC0130), and the Fundamental Research Funds for the Central Universities (PA2025GDGP0026, JZ2024YQTD0600 for Y.-M.L., JZ2025HGTB0183 for G.-C.C.).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.5c01395.

  • Routine reagents and instruments, experimental procedures, and source experimental data (PDF)

§.

T.C., N.L., and J.S. contributed equally. T.C. performed the experiments and wrote the manuscript. N.L. designed the experiments, contributed to data analysis. J.S. and J. L. participated in data analysis. Y.L. and G.C. conceived and designed the experiments and revised the manuscript. All the authors read and approved the final manuscript.

The authors declare no competing financial interest.

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