Site-specific PEGylation of Recombinant Protein SAC-TRAIL and Characterization of the Effect on Antitumor Activity

Site-specific PEGylation of Recombinant Protein SAC-TRAIL and Characterization of the Effect on Antitumor Activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Site-specific PEGylation of Recombinant Protein SAC-TRAIL and Characterization of the Effect on Antitumor Activity Shuting Pan, Yuguo Dong, Xuedong Wang, Yuhong Ren, Zebo Xiu, Jian Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5885641/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Mar, 2025 Read the published version in Molecular Biology Reports → Version 1 posted 7 You are reading this latest preprint version Abstract Background Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent with selective cytotoxicity across a broad spectrum of tumor cell lines. In previous studies, we engineered a recombinant protein drug, SAC-TRAIL, which significantly enhanced the antitumor activity of TRAIL without exhibiting toxicity to normal cells. However, its application in cancer therapy is restricted due to poor resistance to proteolytic degradation and a limited in vivo half-life. Methods and Results To address these limitations, we designed a site-specific PEGylation method by conjugating methoxy-polyethylene glycol maleimide (mPEG-MAL) to the thiol group of specific cysteine residues on SAC-TRAIL. In this study, we optimized the PEGylation conditions for SAC-TRAIL, evaluated the in vitro activity and stability of mPEG-MAL-SAC-TRAIL, and conducted in vivo studies to assess its antitumor efficacy. It was shown that approximately 95% of SAC-TRAIL was PEGylated by mPEG-MAL within 30 minutes, exhibiting improved in vitro stability and antitumor activity. Furthermore, mPEG-MAL-SAC-TRAIL demonstrated enhanced anti-tumor activity and stability in an animal tumor model. Conclusions In summary, site-specific PEGylation at Cys-SH residues offers a promising strategy for extending the effective duration of SAC-TRAIL. Recombinant protein Site-specific modification mPEG-MAL Stability Cancer therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) exhibits inhibitory effects on a wide range of cancer cells without toxic side effects on normal cells [ 1 ]. Although TRAIL shows an attractive prospect for cancer therapy, it induces drug resistance in tumor cells with high NF-κB activity [ 2 ]. SAC-TRAIL is a fusion protein composed of TRAIL and the selective for apoptosis of cancer cells domain (SAC) of the prostate apoptosis response-4 (Par-4), linked by a flexible linker (G 4 S) 3 [ 3 ]. Studies have shown that TRAIL binds to death receptors DR4 and DR5, inducing apoptosis in cancer cells through receptor-mediated internalization, while the SAC domain interacts with the cell surface receptor GRP78 to activate the caspase cascade and inhibit the activity of NF-κB, thereby achieving targeted inhibition [ 4 , 5 ]. Furthermore, both SAC and TRAIL have been reported to be non-cytotoxic to normal cells, making SAC-TRAIL a highly promising recombinant protein drug [ 6 ]. Although recombinant protein drugs offer advantages such as high specificity and well-defined biological functions, they also present challenges, including a short serum half-life in vivo , suboptimal pharmacokinetics, and immunogenicity. These issues necessitate frequent dosing in clinical treatments, increasing patient discomfort and treatment costs [ 8 – 10 ]. Protein PEGylation has become one of the more established modification methods in the field of biopharmaceuticals. To date, the FDA has approved more than 20 PEGylated drugs, further demonstrating the safety of this technology [ 11 , 12 ]. The advantages of PEGylation include enhancing protein solubility, reducing immunogenicity, and protecting protein drugs from proteolytic enzymes through shielding effects. Additionally, PEGylation increases the molecular weight (Mw) of protein drugs, reducing glomerular filtration and thereby extending their serum half-life in vivo [ 13 ]. PEGylation strategies generally include non-site-specific and site-specific modifications. While random modification targeting the ε-NH₂ or α-NH₂ groups of lysine residues can achieve PEGylation and partially improve drug stability, the non-site-specific approach yields a mixture of PEGylated isomers, complicating purification process. Moreover, the spatial shielding effect of PEG at the receptor-binding sites of protein drug can impact its biological activity [ 14 ]. Therefore, selectively performing site-specific PEGylation that away from protein bioactive sites and non-conserved regions is a preferred approach [ 15 ]. Site-specific modifications primarily include amino PEGylation, as well as N-terminal and C-terminal PEGylation. Notably, among various site-specific modification strategies, most researchers employ activated acyl N-hydroxysuccinimide (NHS) ester reagents to form amide bonds with lysine residues for protein functionalization. However, this method requires more complex reaction conditions and can still reduce the biological activity of proteins [ 8 ]. Site-specific PEGylation of recombinant protein drugs via cysteine-thiol modification enables more precise control over conjugation sites. This method not only preserves the stability of the protein secondary structure but also simplifies the purification process and improves modification efficiency. Due to the high nucleophilicity and low abundance of thiol groups, and the fact that cysteine residues on proteins are much less than lysine residues, these factors are important for site-specific modifications [ 11 , 16 ]. In recent studies, the methoxy-polyethylene glycol maleimide (mPEG-MAL) are commonly used as the site-specific modification material, because its maleimide group will rapidly react with thiol groups under reducing conditions to form stable thioether bonds, allowing for the preparation of PEGylated recombinant protein drugs [ 17 ]. The TRAIL, which contains amino acid residues 114–281, preserves the original anticancer activity of the intact protein. The C230 of TRAIL, serving as a trimer chelation point, avoids potential issues such as structural disruption and blockage of active sites, making it a naturally suitable site for specific modifications [ 1 , 17 ]. The core domain of Par-4 (SAC) consists of 59 amino acids, and analysis of the evolutionary patterns and conservation levels of homologous proteins using the bioinformatics tool Confurf ( https://consurf.tau.ac.il/consurf_index.php ) indicates that C29 is a non-conserved site and does not participate in protein-protein interactions, making it a potential candidate for PEGylation [ 7 , 18 ]. In this study, the mPEG-MAL was selected for site-specific modification of the fusion protein SAC-TRAIL to improve the biological stability. The optimal modification conditions for PEGylation were investigated. Furthermore, the biological stability, in vitro and in vivo antitumor activity of the PEGylated SAC-TRAIL was evaluated. This would provide a promising candidate for the treatment of cancer. Materials and Methods Materials Plasmid containing the gene encoding for SAC-TRAIL was constructed according to the method described by Dong et al [ 3 ]. Nickel affinity chromatography column was obtained from Merck (Germany). Methoxy-polyethylene glycol maleimide (mPEG-MAL, 10 kDa) was purchased from Tanshtech (Guangzhou, China). The human ovarian carcinoma cells SK-OV-3 and human embryonic kidney cells HEK-293 were obtained from cell bank of Chinese Academy of Science (Shanghai, China). Medium supplemented with 10% fetal bovine serum (FBS, Gibco) was purchased from Rainbio (Shanghai, China). Cell Counting Kit-8 was from Yeasen (Shanghai, China). Hoechst 33342 was purchased from Maokangbio (Shanghai, China). Annexin V-FITC Apoptosis Detection Kit was from Solarbio (Beijing, China). Structural Simulation The structural model of the recombinant protein SAC-TRAIL with the highest confidence was selected by submitting the amino acid sequence of the target protein to the AlphaFold 3. Model quality was checked by SAVES 6.0. The accuracy of the model was subsequently determined by comparing the errat scores with the assessed values (Fig. S1 ). PyMOL was used to visualize the secondary structure of the protein and confirm the feasibility of PEGylation modification sites. This ensures that site-specific PEGylation of Cys-SH minimizes the impact on the biological activity of SAC-TRAIL. Optimization of Conditions for PEGylation In an oxidizing environment, sulfur atoms (-S-) on cysteines are easy to form disulfide bonds (-S-S-) with each other. To prepare mPEG-MAL-SAC-TRAIL, recombinant protein SAC-TRAIL that dissolved in 20 mM phosphate buffer (PBS, pH 7.4) were treated with the reducing agent tris-(2-carbonyloxyethyl)-phosphine hydrochloride (TCEP-HCl) with a molar ratio of 10:1 for 4 h at 18°C to disrupt the disulfide bonds. The efficiency of polyethylene glycol modification was mainly related to temperature, pH, molar amount of PEG and incubation time. The modification conditions were optimized by the one-factor controlled variable method to achieve maximum polyethylene glycolization, reduce by-products and facilitate subsequent purification. The protein samples obtained under different conditions were subjected to SDS-PAGE to compare the degree of polyethylene glycol modification. Preparation and Purification of mPEG-MAL-SAC-TRAIL The gene encoding SAC-TRAIL was constructed on plasmid pET-28a(+) and expressed in E. coli BL21(DE3). When the OD 600 of bacterial culture reached 0.6–0.8, 1 mM IPTG was added to induce protein expression. Bacteria were collected after 16–18 h and resuspended in lysis buffer (20 mM PBS, pH 7.4). After 15 min of sonication, the organisms were broken and the supernatant was collected by centrifugation for 15 min (10,000 ×g). The target protein was purified by AKTA protein purifier. The lysate supernatant was up-sampled on a Ni-NTA affinity chromatography column and eluted with 20 mM PBS buffer containing different concentrations of imidazole. The purity and protein molecular weights were analysed by SDS-PAGE. Bradford assay was used to determine the concentration of the purified proteins. SAC-TRAIL was modified with mPEG-MAL through the optimal modification conditions screened. Subsequently, purification was performed using ion exchange chromatography, and 20 mM PB buffer containing different concentrations of NaCl was used for elution buffer to remove unreacted mPEG-MAL and proteins that had not been successfully modified. Proteolysis Assay Trypsin and pepsin were diluted in 0.1 mol/L Tris-HCl (pH 8.0) and 0.1 mol/L HCl buffer, respectively. Trypsin and pepsin were added to the purified SAC-TRAIL and mPEG-MAL-SAC-TRAIL at a final concentration of 0.1 nM. After incubation at 37 ℃ for different times, the termination solution was added. The termination solution of trypsin was 0.1% TFA and the termination solution of pepsin was Tris-HCl buffer (pH 8.0). Cell Proliferation Assay The human ovarian cancer cells SK-OV-3 that were stably adherent were collected and washed by PBS for twice. The cells were seeded in 96-well plates at a density of 1 × 10 5 /well and incubated for 24 h at 37 ℃ with 5% CO 2 . Cells were treated with different concentrations of SAC-TRAIL or mPEG-MAL-SAC-TRAIL for 72 h. Subsequently, 10 µL of CCK-8 solution was added to each well, and the cells were incubated at 37 ℃ for 1 h. Absorbance was measured at 450 nm by Microplate Reader. Data were fitted using non-linear regression in GraphPad Prism 9.0 software to calculate IC 50 . Flow Cytometry Assay The apoptosis rates of SAC-TRAIL and mPEG-MAL-SAC-TRAIL were examined by Annexin V-FITC apoptosis detection kit (Solarbio, China). The SK-OV-3 and HEK-293 cells were washed twice with PBS and seeded in 6-well plates at a density of 2×10 5 cells/well and cultured overnight for 24 h. Subsequently, different concentrations of protein drugs were added and incubated for 48 h. Cells were collected by centrifugation at low temperature and washed with pre-cooled PBS, and resuspended by 100 µL of binding buffer. Then, 5 µL of FITC and 5 µL of PI were sequentially added for staining, and the cells were incubated in low-temperature for 10 min in the dark. Finally, 500 µL of PBS buffer was added, and apoptosis was immediately detected on a flow cytometer (Beckman Coulter) and analyzed on Flow Jo software. Immunofluorescence Assay Immunofluorescence assay was used for co-localization detection of the modified recombinant proteins to confirm the target binding efficiency. The purified protein drug was dialyzed in a cross-linking buffer (sodium carbonate buffer, pH 9.0) at 4°C for 3 times. Then, the cross-linking reaction was performed in the ratio of 1 mg protein with 15 µg FITC in the dark for 12 h. Finally, the reaction was terminated with NH 4 Cl at a final concentration of 50 mM. The SK-OV-3 and HEK-293 cells were seeded in laser confocal dishes at a density of 1 × 10 5 cells/mL and incubated overnight, then 5 nM of protein drug was added. After incubating for 12 h, the nuclei were stained at 37°C using Hoechst 33342 (10 µg/mL) and PBS was added to maintain osmolarity. The cells were scanned and photographed under laser confocal microscope. In vivo Antitumor Activity of mPEG-MAL-SAC-TRAIL The human ovarian cancer cells SK-OV-3 were used to establish tumor xenograft mouse model. SK-OV-3 cells were injected subcutaneously into the right abdomen of female Balb/c nude mice (6 weeks old, 1 × 10 8 cells per mouse) and randomly divided into 3 groups (saline, SAC-TRAIL and mPEG-MAL-SAC-TRAIL, six mice per group). On days 4, 7, 11, 14, 18, 21, 25, 28, 32, 35, and 39 after inoculation, each group of mice was injected intravenously with a 20 mg/kg dose of SAC-TRAIL or mPEG-MAL-SAC-TRAIL or an equal volume of saline. Tumor volumes were measured and recorded daily by measuring the longitudinal (L) and transverse (W) diameters of the tumor grafts and calculating the tumor volume (V) by the following formula: V = (L × W 2 )/2 . Meanwhile, the body weights of the mice were monitored daily. Statistical Analyses All data were measured by at least three independent experiments. The experimental data were statistically processed by GraphPad Prism 9.0 software and the data results were expressed as mean ± standard error (mean ± SE). Comparisons between multiple groups were performed using one-way ANOVA and Dunnett's test. Data were statistically significant when P < 0.05. Results Preparation and Characterization of mPEG-MAL-SAC-TRAIL Due to the TRAIL 114 − 281 maintains the original activity of full-length TRAIL, we previously constructed a fusion protein SAC-TRAIL composed of TRAIL 114 − 281 and SAC that linked by a flexible linker (G 4 S) 3 . There is only one cysteine site (C230) in TRAIL 114 − 281 , which makes TRAIL chelated to form a trimer and is not involved in protein interactions [ 16 ]. Meanwhile, the SAC contains 59 amino acids, and the C29 is a non-conserved site based on analysis of the evolutionary patterns and conservation levels of homologous proteins, which facilitates for the site-directed PEGylation of recombinant protein. To analyze the feasibility of polyethylene glycolization, we visualized the structure model of SAC-TRAIL that predicted by AlphaFold 3 at the highest confidence interval (Fig. 1 A). Structural simulations showed that both cysteine sites (C29 and C230) on SAC-TRAIL are exposed on the surface of the fusion protein, which provides the possibility of the modification reaction. In addition, the reaction formula for the polyethylene glycolization of SAC-TRAIL was briefly plotted (Fig. 1 B and C). The purified recombinant protein SAC-TRAIL was analyzed by SDS-PAGE, and it was shown that the apparent molecular weight was approximately 33 kDa, which was consistent with the theoretical molecular weight (Fig. S2). Since cysteine residues are easy to form disulfide bonds with each other, SAC-TRAIL was treated with TCEP-HCl in 20 mM PBS buffer before the preparation of mPEG-MAL-SAC-TRAIL. Subsequently, the modification reactions were carried out at different temperatures, pH, PEG molar amounts and incubation times, respectively. To determine the optimal reaction temperature, the PEGylation was performed at pH 7.4 for 1 h under the molar ratio of mPEG-MAL to SAC-TRAIL at 50:1. The SDS-PAGE analysis showed that the highest PEGylation degree was happened at 25 ℃ (Fig. 2 A). To test the effect of pH on PEGylation, the proteins were reacted at 25 ℃ for 1 h with a 50-fold molar excess of mPEG-MAL. The results showed that although the degree of modification is better at pH 9.0, there is a noticeable white flocculent precipitation. We speculated that pH 9.0 is near the isoelectric point of SAC-TRAIL, which leading to protein precipitation. Therefore, it is better to perform the modification reaction at pH 7.4 (Fig. 2 B). In addition, the modification reaction was carried out at pH 7.4 and 25 ℃ with different molar excesses of mPEG-MAL, and it could be found that the best modification degree of SAC-TRAIL was obtained when the molar ratio of mPEG-MAL to SAC-TRAIL was 50:1 (Fig. 2 C). Meanwhile, it was experimentally verified that the PEGylation degree could not be further improved by increase the amount of mPEG-MAL, and the higher amount of unreacted mPEG-MAL was unfavorable for the subsequent purification. Finally, to determine the optimal incubation time for the modification reaction, SAC-TRAIL was subjected to pH 7.4, 25 ℃, and a 50-fold molar excess of mPEG-MAL. The results showed that the PEGylation could be completed at 30 min (Fig. 2 D). In addition, the degree of cross-linking does not increase with time, but rather affects the biostability of the modified protein. Therefore, we showed that the optimal reaction conditions for the polyethylene glycolization of SAC-TRAIL were pH 7.4, 25 ℃, molar ratio of mPEG-MAL to protein at 50:1, and reaction for 1 h. After the modification, the unreacted mPEG-MAL and the SAC-TRAIL that could not be PEGylated were removed by a weak cation exchange column (CM FF) (Fig. 2 E). The in vitro Antitumor Activity of mPEG-MAL-SAC-TRAIL In order to evaluate the in vitro anti-tumor activity of the PEGlyated protein drugs, the cell proliferation inhibition effect against human ovarian cancer cells SK-OV-3 was examined using the CCK-8 assay. The results showed that mPEG-MAL-SAC-TRAIL (IC 50 2.30 nM) exhibited better cytotoxicity than SAC-TRAIL (IC 50 3.53 nM) (Fig. 3 A). However, in normal human embryonic kidney cells HEK-293, SAC-TRAIL and mPEG-MAL-SAC-TRAIL showed little or no toxic effects on the cells. Subsequently, the ability of SAC-TRAIL and mPEG-MAL-SAC-TRAIL to induce apoptosis in cancer cells was detected by flow cytometry. After incubating with cancer cells for 48 h, 10 nM of SAC-TRAIL induced apoptosis of 53.36% SK-OV-3 cells, while the same concentration of mPEG-MAL-SAC-TRAIL induced apoptosis of 61.23% SK-OV-3 cells (Fig. 3 B and Fig. S3). It can be seen that mPEG-MAL-SAC-TRAIL had a better ability to induce apoptosis of cancer cells than SAC-TRAIL, and both of them could not induce apoptosis in normal cells HEK-293. Combined with the results of CCK-8, it is indicated that the polyethylene glycol modification has no side effect on the original anticancer activity of SAC-TRAIL. Furthermore, the effect of PEGylation on the recognization and combination of SAC-TRAIL with cancer cells can be further verified by confocal microscopy photography, which visualizes the targeting ability of the protein drug on cancer cells (Fig. 3 C). The results showed that polyethylene glycolized SAC-TRAIL does not affect its cell-penetrating ability, which subsequently induces apoptosis of tumor cells. Moreover, there was no green fluorescence observed in HEK-293 cells, which indicated that mPEG-MAL-SAC-TRAIL had no targeted killing effect on normal cells. In addition, the C29 and C230 sites of SAC-TRAIL was mutated to serine that with the same polarity and structure as cysteine, respectively, to assess the importance of cysteine on the bioactivity of SAC-TRAIL. The IC 50 of SAC-TRAIL (C29S) and SAC-TRAIL (C230S) against SK-OV-3 cells was 8.93nM and 6.57nM, respectively, which was significantly reduced compared with SAC-TRAIL (IC 50 3.53 nM) (Fig. S4). The results showed that the C29 and C230 were pivotal for the antitumor effect and site-specific modification of SAC-TRAIL. PEGylation Improved the Stability of SAC-TRAIL The stability of SAC-TRAIL and mPEG-MAL-SAC-TRAIL in vitro can be evaluated by thermal stability, pH stability, and resistance to enzymatic degradation [ 19 ]. For the evaluation of thermal stability, the SAC-TRAIL and mPEG-MAL-SAC-TRAIL were treated at different temperatures for 2 h and then incubated with SK-OV-3 cells, respectively. With the increase of temperature, the IC 50 of mPEG-MAL-SAC-TRAIL were lower than that of SAC-TRAIL and could be maintained at a more stable level, indicating that polyethylene glycolization enhanced the thermal stability of the protein drug SAC-TRAIL (Fig. 4A). Furthermore, SAC-TRAIL and mPEG-MAL-SAC-TRAIL were incubated in different pH for 2 h. The proliferation inhibitory activity of mPEG-MAL-SAC-TRAIL was higher than that of SAC-TRAIL at each pH value, and the mPEG-MAL-SAC-TRAIL had the optimal activity at pH 7.4 (Fig. 4B). Therefore, the PEGylated SAC-TRAIL had better tolerance to changes in pH. For the evaluation of the enzymatic resistance of protein drugs after polyethylene glycolization, two enzymes, trypsin and pepsin, were selected [ 20 ]. The results showed that the bioactivity of SAC-TRAIL was significantly affected with the prolongation of the enzymatic digestion time. However, mPEG-MAL-SAC-TRAIL was tolerant to enzymatic digestion and maintained relatively higher biological activity after 2 h of enzymatic treatment (Fig. 4C and C). The SDS-PAGE analysis showed that the amount of mPEG-MAL-SAC-TRAIL was maintained stable even incubation for 2 h, while SAC-TRAIL showed an obvious degradation (Fig. S5). In summary, the mPEG-MAL-SAC-TRAIL has better biostability compared with SAC-TRAIL, and the polyethylene glycolization is of remarkable significance for the long-lasting modification of protein drugs. Figure 4 Stability of mPEG-MAL-SAC-TRAIL in vitro . ( A ) Thermal stability. SAC-TRAIL and mPEG-MAL-SAC-TRAIL were treated at 37 ℃, 45 ℃, 55 ℃ and 60 ℃ for 2 h and then incubated with SK-OV-3 cells for 72 h, respectively. ( B ) Tolerance to changes in pH. SAC-TRAIL and mPEG-MAL-SAC-TRAIL were treated at pH 6, 7, 7.4, 8, and 9 for 2 h and then incubated with SK-OV-3 cells for 72 h, respectively. ( C ) Resistance to trypsin hydrolysis. SAC-TRAIL and mPEG-MAL-SAC-TRAIL were treated with trypsin for 30, 60, 90, and 120 min, respectively, and then incubated with SK-OV-3 cells for 72 h. ( D ) Resistance to pepsin hydrolysis. SAC-TRAIL and mPEG-MAL-SAC-TRAIL were treated with pepsin for 30, 60, 90, and 120 min, respectively, and then incubated with SK-OV-3 cells for 72 h. **, P < 0.01, ***, P < 0.001, ****, P < 0.0001 Antitumor Activity of mPEG-MAL-SAC-TRAIL in vivo To evaluate the antitumor activity of SAC-TRAIL and mPEG-MAL-SAC-TRAIL in vivo , the SK-OV-3 xenograft mouse models were constructed. As shown in Fig. 5A, mPEG-MAL-SAC-TRAIL significantly inhibited the growth of SK-OV-3 cells compared with the control group and SAC-TRAIL-treated group. The mice which were injected with mPEG-MAL-SAC-TRAIL showed almost absolute tumor growth inhibition after 11 days. Compared with the control group, mPEG-MAL-SAC-TRAIL had less effect on the growth of mice, which would continue to grow after dosing, while the body weight of the other groups was basically unchanged (Fig. 5B). Figure 5 Antitumor activity of mPEG-MAL-SAC-TRAIL in vivo . ( A ) Tumor growth was suppressed by SAC-TRAIL or mPEG-MAL-SAC-TRAIL (20 mg/kg). ( B ) Body weight of each mouse in three groups was measured. Results are expressed as mean ± SD. (n = 6). Discussion The recombinant protein SAC-TRAIL is a fusion of TRAIL, a member of the tumor necrosis factor families, and SAC, the antitumor structural domain of the Par-4 protein, which has broad-spectrum antitumor activity and is a highly promising protein drug [ 1 , 7 ]. Previous studies have shown that although SAC-TRAIL exhibits higher tumor targeting ability and cytotoxicity in vitro , its short half-life and susceptibility to protease hydrolysis in vivo greatly limit its clinical application [ 21 ]. Polyethylene glycol modification has been shown to protect protein drugs from various degradation mechanisms in tissues and cells, and attenuate renal filtration [ 20 , 22 – 24 ]. Although non-site-specific modifications of ε-NH 2 or α-NH 2 are capable of forming some protection for proteins that exhibit stability in vivo , the randomness of the modification sites greatly affects the active site of protein due to the stochastic nature of the modification sites [ 25 ]. In recent years, site-specific modification of proteins has become the mainstream modification method for PEGylation. N-terminal specific polyethylene glycolization has been shown to result in high biological activity of human interleukin-4 (IL-4) and vascular endothelial growth factor 165 (VEGF 165) [ 26 ]. In addition, the sulfhydryl group (-SH) on the side chain of cysteine is a relatively rare and unique reactive group in proteins, which enables highly site-specific modifications and avoids the effects of non-specific modifications on protein structure and function. This specific modification allows precise control of the modification site, thus preserving the activity and function of the protein. In this study, the structure of SAC-TRAIL was simulated by AlphaFold 3, and the spatial conformation showed that both cysteine sites (C29 and C230) are exposed on the surface of the fusion protein, which provides the feasibility of the site-directed modification. We successfully prepared the Cys-SH-specific PEGylated product, mPEG-MAL-SAC-TRAIL, by using the sulfhydryl group in cysteine to react with the maleimide group of mPEG-MAL to generate a thioether. Because of the reaction of the highly reactive maleimide group with the reduced -SH group of cysteine, SAC-TRAIL can be PEGylated quickly and efficiently. Through one-way controlled variable experiments, it was found that SAC-TRAIL could achieve the best modification degree at pH 7.4, 25 ℃, and a 50-fold molar excess of mPEG-MAL, which could modify 95% of SAC-TRAIL in less than 30 min. The antitumor activity of mPEG-MAL-SAC-TRAIL in vitro was verified by CCK-8 and Annexin V/PI staining assay. The results of CCK-8 indicated that the tumor proliferation inhibitory effect of mPEG-MAL-SAC-TRAIL was significantly improved compared with SAC-TRAIL, which suggests that the polyethylene glycol wrapped on the surface of recombinant fusion protein formed a better protective effect on SAC-TRAIL, thus improving the toxicity to tumor cells and enhancing the stability of the drug in vitro . Furthermore, the flow cytometry assay showed that 10 nM of mPEG-MAL-SAC-TRAIL can induce apoptosis in more than 60% of the cancer cells, which has better pro-apoptotic ability than SAC-TRAIL, probably due to the protection of mPEG-MAL that makes the drug less susceptible to proteasomal degradation. The laser confocal assay revealed that PEGylation does not affect the original activity and penetration of SAC-TRAIL toward cancer cells, which further verified the feasibility of PEGylation. Although SAC-TRAIL has shown excellent killing effect on cancer cells in vitro , it is highly susceptible to degradation and inactivation during preparation and storage due to its unstable nature, which significantly limits the application of the drug. Therefore, the biostability of PEGylated SAC-TRAIL under different temperatures, pH and protease were explored. We found that mPEG-MAL-SAC-TRAIL maintained better activity at high temperatures and has better thermal stability. The PEGylated SAC-TRAIL also did not show a significant decrease in activity when the pH was changed. At the same time, in the presence of certain concentrations of trypsin and pepsin, less mPEG-MAL-SAC-TRAIL was degradation compared with SAC-TRAIL, which suggests that polyethylene glycolization is resistant to protease hydrolysis. Therefore, the Cys-SH-specific polyethylene glycolization modification is important to improve the stability of SAC-TRAIL in vitro . Finally, we explored the antitumor activity of PEGylated SAC-TRAIL in vivo by construct the SK-OV-3 xenograft mouse models. It was shown that mPEG-MAL-SAC-TRAIL had a stronger anti-tumor ability in vivo compared to SAC-TRAIL, which further proved the protection of site-directed PEGylation for protein drug. In conclusion, we have developed a site-specific PEGylation method for SAC-TRAIL that showed significant tumor inhibition in vitro and in vivo . The preparation of the PEGylated SAC-TRAIL was optimized, and the temperature, pH and enzymatic stability were improved obviously, which showed enhanced antitumor activity in xenograft mouse models. This study reported on a novel and promising candidate PEGylation strategy for the clinical application of SAC-TRAIL. Declarations Ethics approval and Consent to Participate This study was formally approved by the ethics committee of the East China University of Science and Technology (No. ECUST-2023-050). All experiments on animal were conducted in accordance with the Declaration of Helsinki and that all procedures were carried out according to the guidelines of the National Animal Care and Ethics Institution. Conflict of interest The authors have no financial conflicts of interest to declare. Author Contribution Shuting Pan and Yuguo Dong performed the experiments. Jian Zhang, Xuedong Wang and Yuhong Ren provided the resources. Jian Zhang, Yuhong Ren and Zebo Xiu guided the project and helped with the critical manuscript revisions. All authors reviewed the manuscript. Acknowledgments and Funding This work was supported by the grants from the Natural Science Foundation of Shanghai (22ZR1418300) and National Natural Science Foundation of China (No. 21706072). References Zhang J, Dong WY, Ren YH et al (2022) SAC-TRAIL, a novel anticancer fusion protein: expression, purification, and functional characterization. Appl Microbiol Biotechnol 106:1511–1520 Seo OW, Kim JH, Lee KS et al (2012) Kurarinone promotes TRAIL-induced apoptosis by inhibiting NF-κB-dependent cFLIP expression in HeLa cells. Exp Mol Med 44:653–664 Sarkar S, Jain S, Rai V et al (2015) Plant-derived SAC domain of PAR-4 (Prostate Apoptosis Response 4) exhibits growth inhibitory effects in prostate cancer cells. 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Bioconjug Chem 16:200–207 Batugal T, Pendyala G, Tomasovic L et al (2023) Engineering active lysostaphin variants that incorporate noncanonical amino acids and characterizing the effects of site-specific PEGylation. Biotechnol Bioeng 120:1694–1701 Roberts MJ, Bentley MD, Harris JM (2012) Chemistry for peptide and protein PEGylation. Adv Drug Deliv Rev 54:459–476 Pan LQ, Wang HB, Lai J et al (2013) Site-specific PEGylation of a mutated-cysteine residue and its effect on tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). Biomaterials 34:9115–9123 Ashkenazy H, Abadi S, Martz E et al (2016) ConSurf: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res 44:344–350 Forte N, Livanos M, Miranda E et al (2018) Tuning the hydrolytic stability of next generation maleimide cross-linkers enables access to albumin-antibody fragment conjugates and tri-scFvs. Bioconjug Chem 29:486–492 Santos JHPM, Torres-Obreque KM, Meneguetti GP et al (2018) Protein PEGylation for the design of biobetters: from reaction to purification processes. Brazilian J Pharm Sci 54:e01009 Salphati L, Pang J, Alicke B et al (2024) Preclinical characterization of the absorption and disposition of the brain penetrant PI3K/mTOR inhibitor paxalisib and prediction of its pharmacokinetics and efficacy in human. Xenobiotica 54:64–74 Wang YW, Niu SP, Li BZ et al (2021) Improvement of stability and in vivo antioxidant effect of human glutathione peroxidase mutant by PEGylation. Int J Pharm 609:121152 Meziadi A, Zuberi N, Haan HW et al (2022) Overcoming PEGprotein mutual repulsion to improve the efficiency of PEGylation. Biomacromolecules 23:4948–4956 Deiters A, Cropp TA, Summerer D et al (2004) Site-specific PEGylation of proteins containing unnatural amino acids. Bioorg Med Chem Lett 14:5743–5745 Mishra P, Nayak B, Dey RK (2016) PEGylation in anti-cancer therapy: An overview. Asian J Pharm Sci 11:337–348 Meiners K, Hamm P, Gutmann M et al (2023) Site-specific PEGylation of recombinant tissue-type plasminogen activator. Eur J Pharm Biopharm 192:79–87 Additional Declarations No competing interests reported. Supplementary Files Supplementaryfigure.doc floatimage1.png Cite Share Download PDF Status: Published Journal Publication published 13 Mar, 2025 Read the published version in Molecular Biology Reports → Version 1 posted Editorial decision: Revision requested 07 Feb, 2025 Reviews received at journal 07 Feb, 2025 Reviewers agreed at journal 23 Jan, 2025 Reviewers invited by journal 23 Jan, 2025 Editor assigned by journal 23 Jan, 2025 Submission checks completed at journal 23 Jan, 2025 First submitted to journal 23 Jan, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5885641","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":407142043,"identity":"20911043-f5d1-4e06-a87d-92a5268f8338","order_by":0,"name":"Shuting Pan","email":"","orcid":"","institution":"East China University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Shuting","middleName":"","lastName":"Pan","suffix":""},{"id":407142044,"identity":"ee8da208-5a6d-4fcf-ad29-58eede7efb82","order_by":1,"name":"Yuguo Dong","email":"","orcid":"","institution":"East China University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yuguo","middleName":"","lastName":"Dong","suffix":""},{"id":407142045,"identity":"bd015d55-2d71-4cdb-b8e2-9e13c45a87b6","order_by":2,"name":"Xuedong Wang","email":"","orcid":"","institution":"East China University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xuedong","middleName":"","lastName":"Wang","suffix":""},{"id":407142046,"identity":"5d11d150-1404-4787-94e9-ed9161e1510b","order_by":3,"name":"Yuhong Ren","email":"","orcid":"","institution":"New World Institute of Biotechnology, East China University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yuhong","middleName":"","lastName":"Ren","suffix":""},{"id":407142047,"identity":"1bc3f117-1934-4ac1-a1ad-f3b6e3f37919","order_by":4,"name":"Zebo Xiu","email":"","orcid":"","institution":"Liaocheng People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zebo","middleName":"","lastName":"Xiu","suffix":""},{"id":407142048,"identity":"1446da40-9881-4aef-993a-0cf4cd137b29","order_by":5,"name":"Jian Zhang","email":"data:image/png;base64,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","orcid":"","institution":"East China University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Jian","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-01-23 06:38:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5885641/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5885641/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11033-025-10412-7","type":"published","date":"2025-03-13T15:57:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":74955886,"identity":"65962ceb-609f-4645-87cb-5534c42da929","added_by":"auto","created_at":"2025-01-28 17:30:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":341270,"visible":true,"origin":"","legend":"\u003cp\u003eThe Cys-SH site-specific PEGylation of SAC-TRAIL. (\u003cstrong\u003eA\u003c/strong\u003e) AlphaFold 3 was used to simulate the structure of SAC-TRAIL. (\u003cstrong\u003eB\u003c/strong\u003e) Two cysteinesites on the SAC-TRAIL that bind mPEG-MAL were identified using PyMOL. (\u003cstrong\u003eC\u003c/strong\u003e) The Cys-SH site-specific PEGylation of the fusion protein was accomplished via the conjugation of SAC-TRAIL with mPEG-MAL.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5885641/v1/a8383da03027a47e710db9e7.png"},{"id":74955900,"identity":"85b5d1ed-dd39-4840-8792-6322656b5349","added_by":"auto","created_at":"2025-01-28 17:30:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":393962,"visible":true,"origin":"","legend":"\u003cp\u003eSDS-PAGE analysis of the reaction conditions for PEGylated SAC-TRAIL. (\u003cstrong\u003eA\u003c/strong\u003e) The PEGylation of SAC-TRAIL at different temperatures. Lane\u003cem\u003e \u003c/em\u003eM: protein marker, lane 1: SAC-TRAIL, lanes 2-4: the reaction temperatures were 4 ℃, 18 ℃, and 25 ℃, respectively. (\u003cstrong\u003eB\u003c/strong\u003e) The PEGylation of SAC-TRAIL at different pH. Lane\u003cem\u003e \u003c/em\u003eM: protein marker, lane 1: SAC-TRAIL, lanes 2-6: the reaction pH were 6, 7, 7.4, 8, 9, respectively. (\u003cstrong\u003eC\u003c/strong\u003e) The PEGylation of SAC-TRAIL at different mPEG-MAL molar amounts. Lane\u003cem\u003e \u003c/em\u003eM: protein marker, lane 1: SAC-TRAIL, lanes 2-5: the reaction molar ratios of mPEG-MAL to protein were 5:1, 10:1, 30:1, and 50:1, respectively. (\u003cstrong\u003eD\u003c/strong\u003e) The PEGylation of SAC-TRAIL at different incubation times. Lane\u003cem\u003e \u003c/em\u003eM: protein marker, lane 1: SAC-TRAIL, lanes 2-6: the reaction times were 30, 60, 120, 240, and 480 min, respectively. (\u003cstrong\u003eE\u003c/strong\u003e) The purification of mPEG-MAL-SAC-TRAIL. Lane\u003cem\u003e \u003c/em\u003eM: protein marker, lanes 2: SAC-TRAIL, lanes 3: blends after polyethylene glycolization, lanes 4-5: the eluates at 200 and 500 mM NaCl.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5885641/v1/4aaee73a2ecd3154d86e6222.png"},{"id":74955906,"identity":"88d0fe0a-9d5d-42ea-bd39-af13cf822f8d","added_by":"auto","created_at":"2025-01-28 17:30:31","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":257691,"visible":true,"origin":"","legend":"\u003cp\u003eAntitumor activities of mPEG-MAL-SAC-TRAIL \u003cem\u003ein vitro\u003c/em\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Proliferation inhibition ability of mPEG-MAL-SAC-TRAIL on tumor cells SK-OV-3 and human embryonic kidney cells HEK-293 was determined by CCK-8 assay. (\u003cstrong\u003eB\u003c/strong\u003e) Flow cytometry was used to detect the ability of mPEG-MAL-SAC-TRAIL to induce apoptosis in tumor cells. (\u003cstrong\u003eC\u003c/strong\u003e) Immunofluorescence assay was performed to detect the targeting ability of mPEG-MAL-SAC-TRAIL. FITC-labeled mPEG-MAL-SAC-TRAIL were incubated with SK-OV-3 and HEK-293 cells, respectively, for 12 h. Cell nuclei were stained with Hoechst 33342 for 20 min. Confocal images were captured at × 60 magnification. ****, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001, *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5885641/v1/80c9e5ec04696bd721b6bea2.jpeg"},{"id":74955920,"identity":"7c61e897-c1bf-4264-b3d6-40fc01df7ee5","added_by":"auto","created_at":"2025-01-28 17:30:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":294470,"visible":true,"origin":"","legend":"\u003cp\u003eStabilityof mPEG-MAL-SAC-TRAIL \u003cem\u003ein vitro\u003c/em\u003e. (\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eThermal stability. SAC-TRAIL and mPEG-MAL-SAC-TRAIL were treated at 37 ℃, 45 ℃, 55 ℃ and 60 ℃ for 2 h and then incubated with SK-OV-3 cells for 72 h, respectively. (\u003cstrong\u003eB\u003c/strong\u003e) Tolerance to changes in pH. SAC-TRAIL and mPEG-MAL-SAC-TRAIL were treated at pH 6, 7, 7.4, 8, and 9 for 2 h and then incubated with SK-OV-3 cells for 72 h, respectively. (\u003cstrong\u003eC\u003c/strong\u003e) Resistance to trypsin hydrolysis. SAC-TRAIL and mPEG-MAL-SAC-TRAIL were treated with trypsin for 30, 60, 90, and 120 min, respectively, and then incubated with SK-OV-3 cells for 72 h. (\u003cstrong\u003eD\u003c/strong\u003e) Resistance to pepsin hydrolysis. SAC-TRAIL and mPEG-MAL-SAC-TRAIL were treated with pepsin for 30, 60, 90, and 120 min, respectively, and then incubated with SK-OV-3 cells for 72 h. **, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5885641/v1/f512a365f714750291d44141.png"},{"id":74955922,"identity":"e1863b45-e592-4042-875b-cb08f5f50c1b","added_by":"auto","created_at":"2025-01-28 17:30:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":179610,"visible":true,"origin":"","legend":"\u003cp\u003eAntitumor activityof mPEG-MAL-SAC-TRAIL \u003cem\u003ein vivo\u003c/em\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Tumor growth was suppressed by SAC-TRAIL or mPEG-MAL-SAC-TRAIL (20 mg/kg). (\u003cstrong\u003eB\u003c/strong\u003e) Body weight of each mouse in three groups was measured. Results are expressed as mean ± SD. (n= 6).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5885641/v1/566230c32e74b2761aaf25c7.png"},{"id":78689856,"identity":"aea62b44-2347-4a30-b89b-cd5933ba046d","added_by":"auto","created_at":"2025-03-17 16:13:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2149245,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5885641/v1/148945ca-2424-46b1-990a-94b0bd9c8fa6.pdf"},{"id":74955904,"identity":"23c29cf7-304e-455b-b5fd-a50e438906aa","added_by":"auto","created_at":"2025-01-28 17:30:31","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1113033,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure.doc","url":"https://assets-eu.researchsquare.com/files/rs-5885641/v1/20839d1594b5cdc593f7182c.doc"},{"id":74955903,"identity":"62ed8727-b54f-4212-9e19-538609eb1c97","added_by":"auto","created_at":"2025-01-28 17:30:31","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1216434,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5885641/v1/8b37386411f0870369875acc.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Site-specific PEGylation of Recombinant Protein SAC-TRAIL and Characterization of the Effect on Antitumor Activity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) exhibits inhibitory effects on a wide range of cancer cells without toxic side effects on normal cells [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although TRAIL shows an attractive prospect for cancer therapy, it induces drug resistance in tumor cells with high NF-κB activity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. SAC-TRAIL is a fusion protein composed of TRAIL and the selective for apoptosis of cancer cells domain (SAC) of the prostate apoptosis response-4 (Par-4), linked by a flexible linker (G\u003csub\u003e4\u003c/sub\u003eS)\u003csub\u003e3\u003c/sub\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Studies have shown that TRAIL binds to death receptors DR4 and DR5, inducing apoptosis in cancer cells through receptor-mediated internalization, while the SAC domain interacts with the cell surface receptor GRP78 to activate the caspase cascade and inhibit the activity of NF-κB, thereby achieving targeted inhibition [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Furthermore, both SAC and TRAIL have been reported to be non-cytotoxic to normal cells, making SAC-TRAIL a highly promising recombinant protein drug [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Although recombinant protein drugs offer advantages such as high specificity and well-defined biological functions, they also present challenges, including a short serum half-life \u003cem\u003ein vivo\u003c/em\u003e, suboptimal pharmacokinetics, and immunogenicity. These issues necessitate frequent dosing in clinical treatments, increasing patient discomfort and treatment costs [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eProtein PEGylation has become one of the more established modification methods in the field of biopharmaceuticals. To date, the FDA has approved more than 20 PEGylated drugs, further demonstrating the safety of this technology [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The advantages of PEGylation include enhancing protein solubility, reducing immunogenicity, and protecting protein drugs from proteolytic enzymes through shielding effects. Additionally, PEGylation increases the molecular weight (Mw) of protein drugs, reducing glomerular filtration and thereby extending their serum half-life \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. PEGylation strategies generally include non-site-specific and site-specific modifications. While random modification targeting the ε-NH₂ or α-NH₂ groups of lysine residues can achieve PEGylation and partially improve drug stability, the non-site-specific approach yields a mixture of PEGylated isomers, complicating purification process. Moreover, the spatial shielding effect of PEG at the receptor-binding sites of protein drug can impact its biological activity [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, selectively performing site-specific PEGylation that away from protein bioactive sites and non-conserved regions is a preferred approach [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Site-specific modifications primarily include amino PEGylation, as well as N-terminal and C-terminal PEGylation. Notably, among various site-specific modification strategies, most researchers employ activated acyl N-hydroxysuccinimide (NHS) ester reagents to form amide bonds with lysine residues for protein functionalization. However, this method requires more complex reaction conditions and can still reduce the biological activity of proteins [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSite-specific PEGylation of recombinant protein drugs via cysteine-thiol modification enables more precise control over conjugation sites. This method not only preserves the stability of the protein secondary structure but also simplifies the purification process and improves modification efficiency. Due to the high nucleophilicity and low abundance of thiol groups, and the fact that cysteine residues on proteins are much less than lysine residues, these factors are important for site-specific modifications [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In recent studies, the methoxy-polyethylene glycol maleimide (mPEG-MAL) are commonly used as the site-specific modification material, because its maleimide group will rapidly react with thiol groups under reducing conditions to form stable thioether bonds, allowing for the preparation of PEGylated recombinant protein drugs [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The TRAIL, which contains amino acid residues 114\u0026ndash;281, preserves the original anticancer activity of the intact protein. The C230 of TRAIL, serving as a trimer chelation point, avoids potential issues such as structural disruption and blockage of active sites, making it a naturally suitable site for specific modifications [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The core domain of Par-4 (SAC) consists of 59 amino acids, and analysis of the evolutionary patterns and conservation levels of homologous proteins using the bioinformatics tool Confurf (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://consurf.tau.ac.il/consurf_index.php\u003c/span\u003e\u003cspan address=\"https://consurf.tau.ac.il/consurf_index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) indicates that C29 is a non-conserved site and does not participate in protein-protein interactions, making it a potential candidate for PEGylation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, the mPEG-MAL was selected for site-specific modification of the fusion protein SAC-TRAIL to improve the biological stability. The optimal modification conditions for PEGylation were investigated. Furthermore, the biological stability, \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e antitumor activity of the PEGylated SAC-TRAIL was evaluated. This would provide a promising candidate for the treatment of cancer.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003ePlasmid containing the gene encoding for SAC-TRAIL was constructed according to the method described by Dong et al [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Nickel affinity chromatography column was obtained from Merck (Germany). Methoxy-polyethylene glycol maleimide (mPEG-MAL, 10 kDa) was purchased from Tanshtech (Guangzhou, China). The human ovarian carcinoma cells SK-OV-3 and human embryonic kidney cells HEK-293 were obtained from cell bank of Chinese Academy of Science (Shanghai, China). Medium supplemented with 10% fetal bovine serum (FBS, Gibco) was purchased from Rainbio (Shanghai, China). Cell Counting Kit-8 was from Yeasen (Shanghai, China). Hoechst 33342 was purchased from Maokangbio (Shanghai, China). Annexin V-FITC Apoptosis Detection Kit was from Solarbio (Beijing, China).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStructural Simulation\u003c/h3\u003e\n\u003cp\u003eThe structural model of the recombinant protein SAC-TRAIL with the highest confidence was selected by submitting the amino acid sequence of the target protein to the AlphaFold 3. Model quality was checked by SAVES 6.0. The accuracy of the model was subsequently determined by comparing the errat scores with the assessed values (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). PyMOL was used to visualize the secondary structure of the protein and confirm the feasibility of PEGylation modification sites. This ensures that site-specific PEGylation of Cys-SH minimizes the impact on the biological activity of SAC-TRAIL.\u003c/p\u003e\n\u003ch3\u003eOptimization of Conditions for PEGylation\u003c/h3\u003e\n\u003cp\u003eIn an oxidizing environment, sulfur atoms (-S-) on cysteines are easy to form disulfide bonds (-S-S-) with each other. To prepare mPEG-MAL-SAC-TRAIL, recombinant protein SAC-TRAIL that dissolved in 20 mM phosphate buffer (PBS, pH 7.4) were treated with the reducing agent tris-(2-carbonyloxyethyl)-phosphine hydrochloride (TCEP-HCl) with a molar ratio of 10:1 for 4 h at 18\u0026deg;C to disrupt the disulfide bonds. The efficiency of polyethylene glycol modification was mainly related to temperature, pH, molar amount of PEG and incubation time. The modification conditions were optimized by the one-factor controlled variable method to achieve maximum polyethylene glycolization, reduce by-products and facilitate subsequent purification. The protein samples obtained under different conditions were subjected to SDS-PAGE to compare the degree of polyethylene glycol modification.\u003c/p\u003e\n\u003ch3\u003ePreparation and Purification of mPEG-MAL-SAC-TRAIL\u003c/h3\u003e\n\u003cp\u003eThe gene encoding SAC-TRAIL was constructed on plasmid pET-28a(+) and expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3). When the OD\u003csub\u003e600\u003c/sub\u003e of bacterial culture reached 0.6\u0026ndash;0.8, 1 mM IPTG was added to induce protein expression. Bacteria were collected after 16\u0026ndash;18 h and resuspended in lysis buffer (20 mM PBS, pH 7.4). After 15 min of sonication, the organisms were broken and the supernatant was collected by centrifugation for 15 min (10,000 \u0026times;g). The target protein was purified by AKTA protein purifier. The lysate supernatant was up-sampled on a Ni-NTA affinity chromatography column and eluted with 20 mM PBS buffer containing different concentrations of imidazole. The purity and protein molecular weights were analysed by SDS-PAGE. Bradford assay was used to determine the concentration of the purified proteins. SAC-TRAIL was modified with mPEG-MAL through the optimal modification conditions screened. Subsequently, purification was performed using ion exchange chromatography, and 20 mM PB buffer containing different concentrations of NaCl was used for elution buffer to remove unreacted mPEG-MAL and proteins that had not been successfully modified.\u003c/p\u003e\n\u003ch3\u003eProteolysis Assay\u003c/h3\u003e\n\u003cp\u003eTrypsin and pepsin were diluted in 0.1 mol/L Tris-HCl (pH 8.0) and 0.1 mol/L HCl buffer, respectively. Trypsin and pepsin were added to the purified SAC-TRAIL and mPEG-MAL-SAC-TRAIL at a final concentration of 0.1 nM. After incubation at 37 ℃ for different times, the termination solution was added. The termination solution of trypsin was 0.1% TFA and the termination solution of pepsin was Tris-HCl buffer (pH 8.0).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell Proliferation Assay\u003c/h2\u003e \u003cp\u003eThe human ovarian cancer cells SK-OV-3 that were stably adherent were collected and washed by PBS for twice. The cells were seeded in 96-well plates at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e/well and incubated for 24 h at 37 ℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were treated with different concentrations of SAC-TRAIL or mPEG-MAL-SAC-TRAIL for 72 h. Subsequently, 10 \u0026micro;L of CCK-8 solution was added to each well, and the cells were incubated at 37 ℃ for 1 h. Absorbance was measured at 450 nm by Microplate Reader. Data were fitted using non-linear regression in GraphPad Prism 9.0 software to calculate IC\u003csub\u003e50\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFlow Cytometry Assay\u003c/h3\u003e\n\u003cp\u003eThe apoptosis rates of SAC-TRAIL and mPEG-MAL-SAC-TRAIL were examined by Annexin V-FITC apoptosis detection kit (Solarbio, China). The SK-OV-3 and HEK-293 cells were washed twice with PBS and seeded in 6-well plates at a density of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well and cultured overnight for 24 h. Subsequently, different concentrations of protein drugs were added and incubated for 48 h. Cells were collected by centrifugation at low temperature and washed with pre-cooled PBS, and resuspended by 100 \u0026micro;L of binding buffer. Then, 5 \u0026micro;L of FITC and 5 \u0026micro;L of PI were sequentially added for staining, and the cells were incubated in low-temperature for 10 min in the dark. Finally, 500 \u0026micro;L of PBS buffer was added, and apoptosis was immediately detected on a flow cytometer (Beckman Coulter) and analyzed on Flow Jo software.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence Assay\u003c/h3\u003e\n\u003cp\u003eImmunofluorescence assay was used for co-localization detection of the modified recombinant proteins to confirm the target binding efficiency. The purified protein drug was dialyzed in a cross-linking buffer (sodium carbonate buffer, pH 9.0) at 4\u0026deg;C for 3 times. Then, the cross-linking reaction was performed in the ratio of 1 mg protein with 15 \u0026micro;g FITC in the dark for 12 h. Finally, the reaction was terminated with NH\u003csub\u003e4\u003c/sub\u003eCl at a final concentration of 50 mM.\u003c/p\u003e \u003cp\u003eThe SK-OV-3 and HEK-293 cells were seeded in laser confocal dishes at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL and incubated overnight, then 5 nM of protein drug was added. After incubating for 12 h, the nuclei were stained at 37\u0026deg;C using Hoechst 33342 (10 \u0026micro;g/mL) and PBS was added to maintain osmolarity. The cells were scanned and photographed under laser confocal microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eAntitumor Activity of mPEG-MAL-SAC-TRAIL\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe human ovarian cancer cells SK-OV-3 were used to establish tumor xenograft mouse model. SK-OV-3 cells were injected subcutaneously into the right abdomen of female Balb/c nude mice (6 weeks old, 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e cells per mouse) and randomly divided into 3 groups (saline, SAC-TRAIL and mPEG-MAL-SAC-TRAIL, six mice per group). On days 4, 7, 11, 14, 18, 21, 25, 28, 32, 35, and 39 after inoculation, each group of mice was injected intravenously with a 20 mg/kg dose of SAC-TRAIL or mPEG-MAL-SAC-TRAIL or an equal volume of saline. Tumor volumes were measured and recorded daily by measuring the longitudinal (L) and transverse (W) diameters of the tumor grafts and calculating the tumor volume (V) by the following formula: \u003cem\u003eV = (L \u0026times; W\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)/2\u003c/em\u003e. Meanwhile, the body weights of the mice were monitored daily.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyses\u003c/h2\u003e \u003cp\u003eAll data were measured by at least three independent experiments. The experimental data were statistically processed by GraphPad Prism 9.0 software and the data results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE). Comparisons between multiple groups were performed using one-way ANOVA and Dunnett's test. Data were statistically significant when \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePreparation and Characterization of mPEG-MAL-SAC-TRAIL\u003c/h2\u003e \u003cp\u003eDue to the TRAIL\u003csub\u003e114\u0026thinsp;\u0026minus;\u0026thinsp;281\u003c/sub\u003e maintains the original activity of full-length TRAIL, we previously constructed a fusion protein SAC-TRAIL composed of TRAIL\u003csub\u003e114\u0026thinsp;\u0026minus;\u0026thinsp;281\u003c/sub\u003e and SAC that linked by a flexible linker (G\u003csub\u003e4\u003c/sub\u003eS)\u003csub\u003e3\u003c/sub\u003e. There is only one cysteine site (C230) in TRAIL\u003csub\u003e114\u0026thinsp;\u0026minus;\u0026thinsp;281\u003c/sub\u003e, which makes TRAIL chelated to form a trimer and is not involved in protein interactions [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Meanwhile, the SAC contains 59 amino acids, and the C29 is a non-conserved site based on analysis of the evolutionary patterns and conservation levels of homologous proteins, which facilitates for the site-directed PEGylation of recombinant protein. To analyze the feasibility of polyethylene glycolization, we visualized the structure model of SAC-TRAIL that predicted by AlphaFold 3 at the highest confidence interval (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Structural simulations showed that both cysteine sites (C29 and C230) on SAC-TRAIL are exposed on the surface of the fusion protein, which provides the possibility of the modification reaction. In addition, the reaction formula for the polyethylene glycolization of SAC-TRAIL was briefly plotted (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe purified recombinant protein SAC-TRAIL was analyzed by SDS-PAGE, and it was shown that the apparent molecular weight was approximately 33 kDa, which was consistent with the theoretical molecular weight (Fig. S2). Since cysteine residues are easy to form disulfide bonds with each other, SAC-TRAIL was treated with TCEP-HCl in 20 mM PBS buffer before the preparation of mPEG-MAL-SAC-TRAIL. Subsequently, the modification reactions were carried out at different temperatures, pH, PEG molar amounts and incubation times, respectively. To determine the optimal reaction temperature, the PEGylation was performed at pH 7.4 for 1 h under the molar ratio of mPEG-MAL to SAC-TRAIL at 50:1. The SDS-PAGE analysis showed that the highest PEGylation degree was happened at 25 ℃ (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). To test the effect of pH on PEGylation, the proteins were reacted at 25 ℃ for 1 h with a 50-fold molar excess of mPEG-MAL. The results showed that although the degree of modification is better at pH 9.0, there is a noticeable white flocculent precipitation. We speculated that pH 9.0 is near the isoelectric point of SAC-TRAIL, which leading to protein precipitation. Therefore, it is better to perform the modification reaction at pH 7.4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In addition, the modification reaction was carried out at pH 7.4 and 25 ℃ with different molar excesses of mPEG-MAL, and it could be found that the best modification degree of SAC-TRAIL was obtained when the molar ratio of mPEG-MAL to SAC-TRAIL was 50:1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Meanwhile, it was experimentally verified that the PEGylation degree could not be further improved by increase the amount of mPEG-MAL, and the higher amount of unreacted mPEG-MAL was unfavorable for the subsequent purification. Finally, to determine the optimal incubation time for the modification reaction, SAC-TRAIL was subjected to pH 7.4, 25 ℃, and a 50-fold molar excess of mPEG-MAL. The results showed that the PEGylation could be completed at 30 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In addition, the degree of cross-linking does not increase with time, but rather affects the biostability of the modified protein. Therefore, we showed that the optimal reaction conditions for the polyethylene glycolization of SAC-TRAIL were pH 7.4, 25 ℃, molar ratio of mPEG-MAL to protein at 50:1, and reaction for 1 h. After the modification, the unreacted mPEG-MAL and the SAC-TRAIL that could not be PEGylated were removed by a weak cation exchange column (CM FF) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eAntitumor Activity of mPEG-MAL-SAC-TRAIL\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn order to evaluate the \u003cem\u003ein vitro\u003c/em\u003e anti-tumor activity of the PEGlyated protein drugs, the cell proliferation inhibition effect against human ovarian cancer cells SK-OV-3 was examined using the CCK-8 assay. The results showed that mPEG-MAL-SAC-TRAIL (IC\u003csub\u003e50\u003c/sub\u003e 2.30 nM) exhibited better cytotoxicity than SAC-TRAIL (IC\u003csub\u003e50\u003c/sub\u003e 3.53 nM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, in normal human embryonic kidney cells HEK-293, SAC-TRAIL and mPEG-MAL-SAC-TRAIL showed little or no toxic effects on the cells. Subsequently, the ability of SAC-TRAIL and mPEG-MAL-SAC-TRAIL to induce apoptosis in cancer cells was detected by flow cytometry. After incubating with cancer cells for 48 h, 10 nM of SAC-TRAIL induced apoptosis of 53.36% SK-OV-3 cells, while the same concentration of mPEG-MAL-SAC-TRAIL induced apoptosis of 61.23% SK-OV-3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and Fig. S3). It can be seen that mPEG-MAL-SAC-TRAIL had a better ability to induce apoptosis of cancer cells than SAC-TRAIL, and both of them could not induce apoptosis in normal cells HEK-293. Combined with the results of CCK-8, it is indicated that the polyethylene glycol modification has no side effect on the original anticancer activity of SAC-TRAIL. Furthermore, the effect of PEGylation on the recognization and combination of SAC-TRAIL with cancer cells can be further verified by confocal microscopy photography, which visualizes the targeting ability of the protein drug on cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The results showed that polyethylene glycolized SAC-TRAIL does not affect its cell-penetrating ability, which subsequently induces apoptosis of tumor cells. Moreover, there was no green fluorescence observed in HEK-293 cells, which indicated that mPEG-MAL-SAC-TRAIL had no targeted killing effect on normal cells. In addition, the C29 and C230 sites of SAC-TRAIL was mutated to serine that with the same polarity and structure as cysteine, respectively, to assess the importance of cysteine on the bioactivity of SAC-TRAIL. The IC\u003csub\u003e50\u003c/sub\u003e of SAC-TRAIL (C29S) and SAC-TRAIL (C230S) against SK-OV-3 cells was 8.93nM and 6.57nM, respectively, which was significantly reduced compared with SAC-TRAIL (IC\u003csub\u003e50\u003c/sub\u003e 3.53 nM) (Fig. S4). The results showed that the C29 and C230 were pivotal for the antitumor effect and site-specific modification of SAC-TRAIL.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePEGylation Improved the Stability of SAC-TRAIL\u003c/h2\u003e \u003cp\u003eThe stability of SAC-TRAIL and mPEG-MAL-SAC-TRAIL \u003cem\u003ein vitro\u003c/em\u003e can be evaluated by thermal stability, pH stability, and resistance to enzymatic degradation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For the evaluation of thermal stability, the SAC-TRAIL and mPEG-MAL-SAC-TRAIL were treated at different temperatures for 2 h and then incubated with SK-OV-3 cells, respectively. With the increase of temperature, the IC\u003csub\u003e50\u003c/sub\u003e of mPEG-MAL-SAC-TRAIL were lower than that of SAC-TRAIL and could be maintained at a more stable level, indicating that polyethylene glycolization enhanced the thermal stability of the protein drug SAC-TRAIL (Fig.\u0026nbsp;4A). Furthermore, SAC-TRAIL and mPEG-MAL-SAC-TRAIL were incubated in different pH for 2 h. The proliferation inhibitory activity of mPEG-MAL-SAC-TRAIL was higher than that of SAC-TRAIL at each pH value, and the mPEG-MAL-SAC-TRAIL had the optimal activity at pH 7.4 (Fig.\u0026nbsp;4B). Therefore, the PEGylated SAC-TRAIL had better tolerance to changes in pH. For the evaluation of the enzymatic resistance of protein drugs after polyethylene glycolization, two enzymes, trypsin and pepsin, were selected [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The results showed that the bioactivity of SAC-TRAIL was significantly affected with the prolongation of the enzymatic digestion time. However, mPEG-MAL-SAC-TRAIL was tolerant to enzymatic digestion and maintained relatively higher biological activity after 2 h of enzymatic treatment (Fig.\u0026nbsp;4C and C). The SDS-PAGE analysis showed that the amount of mPEG-MAL-SAC-TRAIL was maintained stable even incubation for 2 h, while SAC-TRAIL showed an obvious degradation (Fig. S5). In summary, the mPEG-MAL-SAC-TRAIL has better biostability compared with SAC-TRAIL, and the polyethylene glycolization is of remarkable significance for the long-lasting modification of protein drugs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;4\u003c/b\u003e Stability of mPEG-MAL-SAC-TRAIL \u003cem\u003ein vitro\u003c/em\u003e. (\u003cb\u003eA\u003c/b\u003e) Thermal stability. SAC-TRAIL and mPEG-MAL-SAC-TRAIL were treated at 37 ℃, 45 ℃, 55 ℃ and 60 ℃ for 2 h and then incubated with SK-OV-3 cells for 72 h, respectively. (\u003cb\u003eB\u003c/b\u003e) Tolerance to changes in pH. SAC-TRAIL and mPEG-MAL-SAC-TRAIL were treated at pH 6, 7, 7.4, 8, and 9 for 2 h and then incubated with SK-OV-3 cells for 72 h, respectively. (\u003cb\u003eC\u003c/b\u003e) Resistance to trypsin hydrolysis. SAC-TRAIL and mPEG-MAL-SAC-TRAIL were treated with trypsin for 30, 60, 90, and 120 min, respectively, and then incubated with SK-OV-3 cells for 72 h. (\u003cb\u003eD\u003c/b\u003e) Resistance to pepsin hydrolysis. SAC-TRAIL and mPEG-MAL-SAC-TRAIL were treated with pepsin for 30, 60, 90, and 120 min, respectively, and then incubated with SK-OV-3 cells for 72 h. **, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntitumor Activity of mPEG-MAL-SAC-TRAIL\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003e To evaluate the antitumor activity of SAC-TRAIL and mPEG-MAL-SAC-TRAIL \u003cem\u003ein vivo\u003c/em\u003e, the SK-OV-3 xenograft mouse models were constructed. As shown in Fig.\u0026nbsp;5A, mPEG-MAL-SAC-TRAIL significantly inhibited the growth of SK-OV-3 cells compared with the control group and SAC-TRAIL-treated group. The mice which were injected with mPEG-MAL-SAC-TRAIL showed almost absolute tumor growth inhibition after 11 days. Compared with the control group, mPEG-MAL-SAC-TRAIL had less effect on the growth of mice, which would continue to grow after dosing, while the body weight of the other groups was basically unchanged (Fig.\u0026nbsp;5B).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;5\u003c/b\u003e Antitumor activity of mPEG-MAL-SAC-TRAIL \u003cem\u003ein vivo\u003c/em\u003e. (\u003cb\u003eA\u003c/b\u003e) Tumor growth was suppressed by SAC-TRAIL or mPEG-MAL-SAC-TRAIL (20 mg/kg). (\u003cb\u003eB\u003c/b\u003e) Body weight of each mouse in three groups was measured. Results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. (n\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe recombinant protein SAC-TRAIL is a fusion of TRAIL, a member of the tumor necrosis factor families, and SAC, the antitumor structural domain of the Par-4 protein, which has broad-spectrum antitumor activity and is a highly promising protein drug [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Previous studies have shown that although SAC-TRAIL exhibits higher tumor targeting ability and cytotoxicity \u003cem\u003ein vitro\u003c/em\u003e, its short half-life and susceptibility to protease hydrolysis \u003cem\u003ein vivo\u003c/em\u003e greatly limit its clinical application [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Polyethylene glycol modification has been shown to protect protein drugs from various degradation mechanisms in tissues and cells, and attenuate renal filtration [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Although non-site-specific modifications of ε-NH\u003csub\u003e2\u003c/sub\u003e or α-NH\u003csub\u003e2\u003c/sub\u003e are capable of forming some protection for proteins that exhibit stability \u003cem\u003ein vivo\u003c/em\u003e, the randomness of the modification sites greatly affects the active site of protein due to the stochastic nature of the modification sites [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In recent years, site-specific modification of proteins has become the mainstream modification method for PEGylation. N-terminal specific polyethylene glycolization has been shown to result in high biological activity of human interleukin-4 (IL-4) and vascular endothelial growth factor 165 (VEGF 165) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In addition, the sulfhydryl group (-SH) on the side chain of cysteine is a relatively rare and unique reactive group in proteins, which enables highly site-specific modifications and avoids the effects of non-specific modifications on protein structure and function. This specific modification allows precise control of the modification site, thus preserving the activity and function of the protein.\u003c/p\u003e \u003cp\u003eIn this study, the structure of SAC-TRAIL was simulated by AlphaFold 3, and the spatial conformation showed that both cysteine sites (C29 and C230) are exposed on the surface of the fusion protein, which provides the feasibility of the site-directed modification. We successfully prepared the Cys-SH-specific PEGylated product, mPEG-MAL-SAC-TRAIL, by using the sulfhydryl group in cysteine to react with the maleimide group of mPEG-MAL to generate a thioether. Because of the reaction of the highly reactive maleimide group with the reduced -SH group of cysteine, SAC-TRAIL can be PEGylated quickly and efficiently. Through one-way controlled variable experiments, it was found that SAC-TRAIL could achieve the best modification degree at pH 7.4, 25 ℃, and a 50-fold molar excess of mPEG-MAL, which could modify 95% of SAC-TRAIL in less than 30 min.\u003c/p\u003e \u003cp\u003eThe antitumor activity of mPEG-MAL-SAC-TRAIL \u003cem\u003ein vitro\u003c/em\u003e was verified by CCK-8 and Annexin V/PI staining assay. The results of CCK-8 indicated that the tumor proliferation inhibitory effect of mPEG-MAL-SAC-TRAIL was significantly improved compared with SAC-TRAIL, which suggests that the polyethylene glycol wrapped on the surface of recombinant fusion protein formed a better protective effect on SAC-TRAIL, thus improving the toxicity to tumor cells and enhancing the stability of the drug \u003cem\u003ein vitro\u003c/em\u003e. Furthermore, the flow cytometry assay showed that 10 nM of mPEG-MAL-SAC-TRAIL can induce apoptosis in more than 60% of the cancer cells, which has better pro-apoptotic ability than SAC-TRAIL, probably due to the protection of mPEG-MAL that makes the drug less susceptible to proteasomal degradation. The laser confocal assay revealed that PEGylation does not affect the original activity and penetration of SAC-TRAIL toward cancer cells, which further verified the feasibility of PEGylation.\u003c/p\u003e \u003cp\u003eAlthough SAC-TRAIL has shown excellent killing effect on cancer cells \u003cem\u003ein vitro\u003c/em\u003e, it is highly susceptible to degradation and inactivation during preparation and storage due to its unstable nature, which significantly limits the application of the drug. Therefore, the biostability of PEGylated SAC-TRAIL under different temperatures, pH and protease were explored. We found that mPEG-MAL-SAC-TRAIL maintained better activity at high temperatures and has better thermal stability. The PEGylated SAC-TRAIL also did not show a significant decrease in activity when the pH was changed. At the same time, in the presence of certain concentrations of trypsin and pepsin, less mPEG-MAL-SAC-TRAIL was degradation compared with SAC-TRAIL, which suggests that polyethylene glycolization is resistant to protease hydrolysis. Therefore, the Cys-SH-specific polyethylene glycolization modification is important to improve the stability of SAC-TRAIL \u003cem\u003ein vitro\u003c/em\u003e. Finally, we explored the antitumor activity of PEGylated SAC-TRAIL \u003cem\u003ein vivo\u003c/em\u003e by construct the SK-OV-3 xenograft mouse models. It was shown that mPEG-MAL-SAC-TRAIL had a stronger anti-tumor ability \u003cem\u003ein vivo\u003c/em\u003e compared to SAC-TRAIL, which further proved the protection of site-directed PEGylation for protein drug.\u003c/p\u003e \u003cp\u003eIn conclusion, we have developed a site-specific PEGylation method for SAC-TRAIL that showed significant tumor inhibition \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. The preparation of the PEGylated SAC-TRAIL was optimized, and the temperature, pH and enzymatic stability were improved obviously, which showed enhanced antitumor activity in xenograft mouse models. This study reported on a novel and promising candidate PEGylation strategy for the clinical application of SAC-TRAIL.\u003c/p\u003e "},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and Consent to Participate\u003c/strong\u003e \u003cp\u003eThis study was formally approved by the ethics committee of the East China University of Science and Technology (No. ECUST-2023-050). All experiments on animal were conducted in accordance with the Declaration of Helsinki and that all procedures were carried out according to the guidelines of the National Animal Care and Ethics Institution.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConflict of interest\u003c/strong\u003e \u003cp\u003eThe authors have no financial conflicts of interest to declare.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eShuting Pan and Yuguo Dong performed the experiments. Jian Zhang, Xuedong Wang and Yuhong Ren provided the resources. Jian Zhang, Yuhong Ren and Zebo Xiu guided the project and helped with the critical manuscript revisions. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments and Funding\u003c/h2\u003e \u003cp\u003eThis work was supported by the grants from the Natural Science Foundation of Shanghai (22ZR1418300) and National Natural Science Foundation of China (No. 21706072).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhang J, Dong WY, Ren YH et al (2022) SAC-TRAIL, a novel anticancer fusion protein: expression, purification, and functional characterization. Appl Microbiol Biotechnol 106:1511\u0026ndash;1520\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeo OW, Kim JH, Lee KS et al (2012) Kurarinone promotes TRAIL-induced apoptosis by inhibiting NF-κB-dependent cFLIP expression in HeLa cells. Exp Mol Med 44:653\u0026ndash;664\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarkar S, Jain S, Rai V et al (2015) Plant-derived SAC domain of PAR-4 (Prostate Apoptosis Response 4) exhibits growth inhibitory effects in prostate cancer cells. Front Plant Sci 6:822\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoornstra JJ, Kleibeuker JH, Geelen CM et al (2003) Expression of TRAIL (TNF-related apoptosis-inducing ligand) and its receptors in normal colonic mucosa, adenomas, and carcinomas. J Pathol 200:327\u0026ndash;335\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpierings DCJ, Vries EGE, Timens W et al (2003) Expression of TRAIL and TRAIL death receptors in stage III non-small cell lung cancer tumors. Clin Cancer Research: Official J Am Association Cancer Res 9:3397\u0026ndash;3405\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeynier S, Kramer M, Ribaux P et al (2015) Role of PAR-4 in ovarian cancer. 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Int J Mol Sci 24:3387\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVanderschuren K, Arranz-Gibert P, Khang M et al (2022) Tuning protein half-life in mouse using sequence-defined biopolymers functionalized with lipids. Proceedings of the National Academy of Sciences 119: e2103099119\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKane BJ, Fettis MM, Farhadi S et al (2021) Site-specific cross-linking of galectin-1 homodimers via poly(ethylene glycol) bismaleimide. Cell Mol Bioeng 14:523\u0026ndash;534\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShang XJ, Brandon C, Brian M et al (2016) Modeling and optimization of protein PEGylation. Ind Eng Chem Res 55:11785\u0026ndash;11794\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQi FB, Hu CY, Yu WL et al (2018) Conjugation with eight-arm PEG markedly improves the in vitro activity and prolongs the blood circulation of staphylokinase. Bioconjug Chem 29:451\u0026ndash;458\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosendahl MS, Doherty DH, Smith DJ et al (2005) A long-acting, highly potent interferon alpha-2 conjugate created using site-specific PEGylation. Bioconjug Chem 16:200\u0026ndash;207\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBatugal T, Pendyala G, Tomasovic L et al (2023) Engineering active lysostaphin variants that incorporate noncanonical amino acids and characterizing the effects of site-specific PEGylation. Biotechnol Bioeng 120:1694\u0026ndash;1701\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoberts MJ, Bentley MD, Harris JM (2012) Chemistry for peptide and protein PEGylation. 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Asian J Pharm Sci 11:337\u0026ndash;348\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeiners K, Hamm P, Gutmann M et al (2023) Site-specific PEGylation of recombinant tissue-type plasminogen activator. Eur J Pharm Biopharm 192:79\u0026ndash;87\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Recombinant protein, Site-specific modification, mPEG-MAL, Stability, Cancer therapy","lastPublishedDoi":"10.21203/rs.3.rs-5885641/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5885641/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent with selective cytotoxicity across a broad spectrum of tumor cell lines. In previous studies, we engineered a recombinant protein drug, SAC-TRAIL, which significantly enhanced the antitumor activity of TRAIL without exhibiting toxicity to normal cells. However, its application in cancer therapy is restricted due to poor resistance to proteolytic degradation and a limited \u003cem\u003ein vivo\u003c/em\u003e half-life.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods and Results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo address these limitations, we designed a site-specific PEGylation method by conjugating methoxy-polyethylene glycol maleimide (mPEG-MAL) to the thiol group of specific cysteine residues on SAC-TRAIL. In this study, we optimized the PEGylation conditions for SAC-TRAIL, evaluated the \u003cem\u003ein vitro\u003c/em\u003e activity and stability of mPEG-MAL-SAC-TRAIL, and conducted \u003cem\u003ein vivo\u003c/em\u003e studies to assess its antitumor efficacy. It was shown that approximately 95% of SAC-TRAIL was PEGylated by mPEG-MAL within 30 minutes, exhibiting improved \u003cem\u003ein vitro\u003c/em\u003e stability and antitumor activity. Furthermore, mPEG-MAL-SAC-TRAIL demonstrated enhanced anti-tumor activity and stability in an animal tumor model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn summary, site-specific PEGylation at Cys-SH residues offers a promising strategy for extending the effective duration of SAC-TRAIL.\u003c/p\u003e","manuscriptTitle":"Site-specific PEGylation of Recombinant Protein SAC-TRAIL and Characterization of the Effect on Antitumor Activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-28 17:30:27","doi":"10.21203/rs.3.rs-5885641/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-07T12:58:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-07T12:28:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"179592553851589591993339468540772793188","date":"2025-01-23T15:39:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-23T12:49:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-23T11:04:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-23T11:03:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2025-01-23T06:26:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"59e1cbe1-4fdb-4a18-806b-44d1d1ac4d9e","owner":[],"postedDate":"January 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-17T16:10:00+00:00","versionOfRecord":{"articleIdentity":"rs-5885641","link":"https://doi.org/10.1007/s11033-025-10412-7","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2025-03-13 15:57:58","publishedOnDateReadable":"March 13th, 2025"},"versionCreatedAt":"2025-01-28 17:30:27","video":"","vorDoi":"10.1007/s11033-025-10412-7","vorDoiUrl":"https://doi.org/10.1007/s11033-025-10412-7","workflowStages":[]},"version":"v1","identity":"rs-5885641","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5885641","identity":"rs-5885641","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}