Conjugation Yield Research Articles

Unveiling the transformative influence of sonochemistry on formation of whey protein isolate and green tea extract (WPI-GTE) conjugates

This study investigated the formation of conjugates between whey protein isolate (WPI) and green tea extract (GTE) using three methods: redox-pair (R), ultrasound-assisted redox-pair (RU), and ultrasonication (UL). Ultrasonication significantly reduced the reaction time for synthesizing WPI-GTE conjugates compared to the standard R method (p < 0.05). The UL methods had the highest conjugate yield determined by polyphenol binding (p < 0.05). Fourier-transform infrared spectroscopy (FTIR) and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the conjugate formation, indicating an increased molecular weight due to protein binding with polyphenols through covalent and non-covalent bonds. Conjugates produced via ultrasonication exhibited enhanced solubility, smaller particle size, better emulsifying capacity, and improved foaming ability compared to those formed using the traditional R method (p < 0.05). However, conjugates from the R method showed higher antioxidant activity, as evidenced by DPPH•and ABTS•+ scavenging activities (p < 0.05). In conclusion, WPI-GTE conjugates created through ultrasonic treatment demonstrate potential as dual-functional ingredients, serving as both antioxidant and emulsifier.

Abstract B030: ABC101-1, a novel microtubule inhibitor antibody-drug conjugate targeting HER2, has potent anti-tumor activity in a breast cancer model

Abstract Background: ABC101-1 is an antibody-drug conjugate (ADC) targeting receptor tyrosine-protein kinase erbB-2, also known as HER2. ABC101-1 consists of a humanized IgG1 monoclonal antibody called trastuzumab conjugated to a microtubulin inhibitor, DM1, through interchain cysteines with a drug-to-antibody ratio (DAR) of 8. Our novel non-cleavable linker platform using maleimide as a connector enabled the construction of homogeneous ABC101-1 with DAR of 8, via a straightforward process resulting in high conjugation yield. Employing non-cleavable linkers may ensure that the cytotoxic drug is delivered stably to tumor cells, enhancing the therapeutic efficacy of ADC. Herein, we describe anti-breast cancer activity through in vitro cytotoxicity and in vivo efficacy, pharmacokinetics, and binding affinity of ABC101-1. Methods: In vitro cytotoxicity of ADC and linker-payload was evaluated by measuring cell viability using Cell Count Kit-8 (CCK8) analysis. In vivo anti-tumor activity was investigated using SKBR3 cell line-derived xenograft (CDX) model. A pharmacokinetic (PK) profile was assessed by performing indirect ELISA to determine concentrations of total mAb in the serum. The binding affinity of ABC101-1 to HER2 protein was assessed using the indirect ELISA measuring the concentrations of total mAb. Results: In this study, ABC101-1 exhibited potent in vitro cytotoxicity with picomolar activity on HER2-positive breast cancer cell line. In the CDX breast cancer model, ABC101-1 led to stronger tumor growth inhibition in both early and late phases than ado-trastuzumab emtansine (DAR 3.5) at the same dose. Also, the favorable pharmacokinetic profile of ABC101-1 was observed, along with the preserved antigen-binding ability upon the conjugation to linker-payload. Conclusions: ABC101-1 displayed preclinical efficacy in in vitro and in vivo studies as well as a favorable PK, thereby suggesting its therapeutic potential as an anti-tumor drug in breast cancer therapy. This study would reveal that our novel linker platform is a promising and efficient strategy to develop effective and homogeneous ADCs with DAR 8. Our finding may support the further pre-clinical evaluation of ABC101-1. Citation Format: Seo Woo Kim, Hana Yu, Geon Ho Lee, Tae Gyu Lim, Minji Jeong, Hyeonjoo Hwang, Jee Eun Moon, Yong Beom Lee, Sung-Ha Jin, Jin Hyun Jeong. ABC101-1, a novel microtubule inhibitor antibody-drug conjugate targeting HER2, has potent anti-tumor activity in a breast cancer model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B030.

Highly efficient site-specific protein modification using tyrosinase from Streptomyces avermitilis: Structural insight

Tyrosinase-mediated protein conjugation has recently drawn attention as a site-specific protein modification tool under mild conditions. However, the tyrosinases reported to date act only on extremely exposed tyrosine residues, which limits where the target tyrosine can be located. Herein, we report a tyrosinase from Streptomyces avermitilis (SaTYR), that exhibits a much higher activity against tyrosine residues on the protein surface than other tyrosinases. We determined the crystal structure of SaTYR and revealed that the enzyme has a relatively flat and shallow substrate-binding pocket to accommodate a protein substrate. We demonstrated SaTYR-mediated fluorescence dye tagging and PEGylation of a surface tyrosine residue that was unreacted by other tyrosinases with an approximately 95.2 % conjugation yield in 1 h. We also present a structural rationale that considers the steric hindrance from adjacent residues and surrounding structures along with the extent of solvent exposure of residues, as necessary when determining the optimal positions for introducing target tyrosine residues in SaTYR-mediated protein modification. The study demonstrated that the novel tyrosinase, SaTYR, extends the scope of tyrosinase-mediated protein modification, and we propose that site-specific tyrosine conjugation using SaTYR is a promising strategy for protein bioconjugation in various applications.

The Novel Tetra-Specific Drug C-192, Conjugated Using UniStac, Alleviates Non-Alcoholic Steatohepatitis in an MCD Diet-Induced Mouse Model.

Non-alcoholic steatohepatitis (NASH) is a complex disease resulting from chronic liver injury associated with obesity, type 2 diabetes, and inflammation. Recently, the importance of developing multi-target drugs as a strategy to address complex diseases such as NASH has been growing; however, their manufacturing processes remain time- and cost-intensive and inefficient. To overcome these limitations, we developed UniStac, a novel enzyme-mediated conjugation platform for multi-specific drug development. UniStac demonstrated high conjugation yields, optimal thermal stabilities, and robust biological activities. We designed a tetra-specific compound, C-192, targeting glucagon-like peptide 1 (GLP-1), glucagon (GCG), fibroblast growth factor 21 (FGF21), and interleukin-1 receptor antagonist (IL-1RA) simultaneously for the treatment of NASH using UniStac. The biological activity and treatment efficacy of C-192 were confirmed both in vitro and in vivo using a methionine-choline-deficient (MCD) diet-induced mouse model. C-192 exhibited profound therapeutic efficacies compared to conventional drugs, including liraglutide and dulaglutide. C-192 significantly improved alanine transaminase levels, triglyceride accumulation, and the non-alcoholic fatty liver disease activity score. In this study, we demonstrated the feasibility of UniStac in creating multi-specific drugs and confirmed the therapeutic potential of C-192, a drug that integrates multiple mechanisms into a single molecule for the treatment of NASH.

Preparation of Self-Assembled, Curcumin-Loaded Nano-Micelles Using Quarternized Chitosan-Vanillin Imine (QCS-Vani Imine) Conjugate and Evaluation of Synergistic Anticancer Effect with Cisplatin.

Nano-micelles are self-assembling colloidal dispersions applied to enhance the anticancer efficacy of chemotherapeutic agents. In this study, the conjugate of quarternized chitosan and vanillin imine (QCS-Vani imine) was synthesized using the reaction of a Schiff base characterized by proton-NMR (1HNMR), UV-Vis spectroscopy, and FT-IR. The critical micelle concentration (CMC), particle size, and zeta potential of the resulting product were determined. The QCS-Vani imine conjugate was used as a carrier for the development of curcumin-loaded nano-micelles, and their entrapment efficiency (%EE), drug-loading capacity (%LC) and in vitro release were investigated using HPLC analysis. Moreover, the nano-micelles containing curcumin were combined with various concentrations of cisplatin and evaluated for a possible anticancer synergistic effect. The anticancer activity was evaluated against lung cancer A549 and mouse fibroblast L929 cell lines. The percent yield (%) of the QCS-Vani imine conjugate was 93.18%. The curcumin-loaded QCS-Vani imine nano-micelles were characterized and found to have a spherical shape (by TEM) with size < 200 nm (by DLS) with high %EE up to 67.61% and %LC up to 6.15 ± 0.41%. The loaded lyophilized powder of the nano-micelles was more stable at 4 °C than at room temperature during 120 days of storage. pH-sensitive release properties were observed to have a higher curcumin release at pH 5.5 (cancer environment) than at pH 7.4 (systemic environment). Curcumin-loaded QCS-Vani imine nano-micelles showed higher cytotoxicity and selectivity toward lung cancer A549 cell lines and exhibited lower toxicity toward the normal cell (H9C2) than pure curcumin. Moreover, the curcumin-loaded QCS-Vani imine nano-micelles exhibited an enhanced property of inducing cell cycle arrest during the S-phase against A549 cells and showed prominently induced apoptosis in lung cancer cells compared to that with curcumin. The co-treatment of cisplatin with curcumin-loaded QCS-Vani imine nano-micelles presented an enhanced anticancer effect, showing 8.66 ± 0.88 μM as the IC50 value, in comparison to the treatment with cisplatin alone (14.22 ± 1.01 μM). These findings suggest that the developed QCS-Vani imine nano-micelle is a potential drug delivery system and could be a promising approach for treating lung cancer in combination with cisplatin.

Comparison in structural, physicochemical and functional properties of sweet potato stems and leaves polysaccharide conjugates from different technologies

In order to better understand the influences of extraction techniques on the yield, characteristics, and bioactivities of polysaccharide conjugates, hot reflux extraction (HRE), ultrasonic-assisted extraction (UAE), microwave-assisted extraction (MAE), complex enzymolysis extraction (CEE), ultra-high pressure extraction (UPE), ultrasonic complex enzymes extraction (UEE) were used to extract sweet potato stems leaves polysaccharide conjugates (SPSPCs), and their physicochemical characteristics, functional properties, antioxidant and hypoglycemic activities were compared. Results showed that compared with HRE conjugate (HR-SPSPC), the yield, content of uronic acid (UAC), total phenol (TPC), total flavonoid (TFC) and sulfate group (SGC), water solubility (WS), percentage of glucuronic acid (GlcA), galacuronic acid (GalA) and galactose (Gal), antioxidant and hypoglycemia activities of UEE polysaccharide conjugates (UE-SPSPC) significant increased, while its molecular weight (Mw), degree of esterification (DE), content of protein (PC) and percentage of glucose (Glc) declined, but monosaccharides and amino acid types, and glycosyl linkages were not much different. Indeed, UE-SPSPC possessed the highest antioxidant activities and hypolipidemic activities among six SPSPCs, which might be due to the high UAC, TPC, TFC, SGC, GlcA, GalA and WS, low Mw, DE and Glc of UE-SPSPC. The results reveal that UEE is an effective extraction and modification technology of polysaccharide conjugates.

Bioconjugation of Carbohydrates to Gelatin Sponges Promoting 3D Cell Cultures.

Gelatin sponges are widely employed as hemostatic agents, and are gaining increasing interest as 3D scaffolds for tissue engineering. To broaden their possible application in the field of tissue engineering, a straightforward synthetic protocol able to anchor the disaccharides, maltose and lactose, for specific cell interactions was developed. A high conjugation yield was confirmed by 1H-NMR and FT-IR spectroscopy, and the morphology of the resulting decorated sponges was characterized by SEM. After the crosslinking reaction, the sponges preserve their porous structure as ascertained by SEM. Finally, HepG2 cells cultured on the decorated gelatin sponges show high viability and significant differences in the cellular morphology as a function of the conjugated disaccharide. More spherical morphologies are observed when cultured on maltose-conjugated gelatin sponges, while a more flattened aspect is discerned when cultured onto lactose-conjugated gelatin sponges. Considering the increasing interest in small-sized carbohydrates as signaling cues on biomaterial surfaces, systematic studies on how small carbohydrates might influence cell adhesion and differentiation processes could take advantage of the described protocol.

Investigation of the effect of chelating nitrogenous bases on click reactions on poly[(methyl acrylate)‐co‐(5‐azido‐1‐pentene)

AbstractClick reactions on poly[(methyl acrylate)‐co‐(5‐azido‐1‐pentene)], synthesized by radical polymerization of methyl acrylate and 1‐bromo‐5‐pentene, and converted into the click‐ready azido form by nucleophilic substitution with sodium azide, were studied. Propargyl benzoate and propargyl acetate were clicked onto the polymeric substrate, with the latter known to be a poor click reactant. The impact of solvents (dimethyl formamide (DMF) and tetrahydrofuran (THF)), catalytic systems (CuSO4/Na ascorbate, [Cu(BPMEN)(ClO4)2]/Na ascorbate, bromotris(triphenylphosphine) copper(I) ([CuBr(PPh3)3])) and the addition of chelating nitrogenous bases (triethylamine (TEA), diisopropylethylamine, N,N,N′,N″,N″‐pentamethyldiethylenetriamine) on the degree of conversion was analyzed. Only reactions catalyzed with [CuBr(PPh3)3] led to high or complete conversions in both solvents. Reactions catalyzed with the pre‐catalyst complexes CuSO4⋅5H2O and [(CuBPMEN)(ClO4)2], which require the in situ reduction of Cu(II) to Cu(I) by sodium ascorbate, yielded incomplete conversions in both solvents. Adding chelating nitrogenous bases, with TEA being the most effective, yielded complete conversions for propargyl benzoate with all three catalytic systems in DMF. Adding TEA to reactions of propargyl acetate in DMF increased conversion by 17.9% for CuSO4⋅5H2O/Na ascorbate and 29.6% for [(CuBPMEN)(ClO4)2]/Na ascorbate. The addition of chelating bases was more effective in DMF than in THF, which was also the less suited solvent in reactions without chelating bases. The results show that click reactions with propargyl reagents that cannot increase the nucleophilicity of the vinylidene‐like β‐carbon through electron conjugation yield lower conversions. The effectiveness of catalytic systems that undergo in situ reductions can be enhanced by nitrogenous chelating bases that stabilize the Cu(I) stage. © 2023 Society of Industrial Chemistry.

Conjugation of Short Oligopeptides to a Second-Generation Polyamidoamine Dendrimer Shows Antibacterial Activity

The growing evolution of bacterial resistance to antibiotics represents a global issue that not only impacts healthcare systems but also political and economic processes. This necessitates the development of novel antibacterial agents. Antimicrobial peptides have shown promise in this regard. Thus, in this study, a new functional polymer was synthesized by joining a short oligopeptide sequence (Phe-Lys-Phe-Leu, FKFL) to the surface of a second-generation polyamidoamine (G2 PAMAM) dendrimer as an antibacterial component. This method of synthesis proved simple and resulted in a high conjugation yield of the product FKFL-G2. To determine its antibacterial potential, FKFL-G2 was subsequently analyzed via mass spectrometry, a cytotoxicity assay, bacterial growth assay, colony-forming unit assay, membrane permeabilization assay, transmission electron microscopy, and biofilm formation assay. FKFL-G2 was found to exhibit low toxicity to noncancerous NIH3T3 cells. Additionally, FKFL-G2 had an antibacterial effect on Escherichia coli and Staphylococcus aureus strains by interacting with and disrupting the bacterial cell membrane. Based on these findings, FKFL-G2 shows promise as a potential antibacterial agent.

Template-Directed Incorporation of Functional Molecules into DNA.

We report a versatile method for the incorporation of functional molecules into oligonucleotides carrying reactive groups by using a template-directed postsynthetic approach in the solution phase. For this purpose, we prepared oligonucleotides carrying an amino group on the backbone by using an acylic threoninol scaffold. The resulting oligonucleotides could be used to introduce almost any molecule carrying aldehyde, which can be, among other things, a metal-binding ligand or a fluorophore. In our study, we incorporated aldehyde-bearing phenanthroline, a metal-binding ligand, into oligonucleotides by template-directed reversible conjugation. We observed that the use of an abasic sugar site instead of a natural nucleobase in the template strand increased the yield of conjugation and induced selective incorporation of the phenanthroline. This method could lead progress in the development of probes for the recognition of abasic regions in duplex DNA. Moreover, template-directed formation of metal ligand-oligonucleotide conjugates might have potential applications in hybrid biocatalysis for enantioselective transformations.

Optimizing and determining the click chemistry mediated Cu-64 radiolabeling and physiochemical characteristics of trastuzumab conjugates

Over the last decade, 64Cu-labeling of monoclonal antibody (mAb) via inverse electron demand Diels-Alder click chemistry (IEDDA) have received much attention. Despite the tetrazine-transcyclooctene (Tz-TCO) click chemistry's convenience and efficiency in mAb labeling, there is limited information about the ideal parameters in the development of click chemistry mediated (radio)immunoconjugates. This encourages us to conduct a systematic optimization while concurrently determining the physiochemical characteristics of the model mAb, trastuzumab, and TCO conjugates. To accomplish this, we investigated a few critical parameters, first, we determined the degree of conjugations with varying molar equivalents (eq.) of TCO (3, 5, 10, and 15 eq.). Through analytical techniques like size exclusion chromatography, dynamic light scattering, and zeta potential, qualitative analysis were performed to determine the purity, degree of aggregation and net charge of the conjugates. We found that as the degree of conjugation increased the purity of intact mAb fraction is compromised and net charge of conjugates became less positive. Next, all trastuzumab-PEG4-TCO conjugates with varying molar ratio and quantity (30, 50, 100, 200, 250 μg) were radiolabeled with 64Cu-NOTA-PEG4-Tz via IEDDA click chemistry and radiochemical yields were determined by radio-thin layer chromatography. The radiochemical yields of trastuzumab conjugates improved with increased amount and molar ratio. Next, we investigated the effect of the radioprotectant ascorbic acid (AA) of varied concentrations (0.25, 0.5, 0.75, 1 mM) on radiochemical yields and subsequent pharmacokinetics. A concentration of 0.25 mM of AA was found to be optimal for click reaction and in vivo biodistribution. Finally, we investigated the indirect influence of bioconjugation buffers on radiochemical yields and biodistribution in NIH3T6.7 tumor models that resulted approximately ∼11 %ID/g tumor uptake.

Combined Self-Assembled iRGD Polymersomes for Effective Targeted siRNA Anti-Tumor Therapy.

iRGD is usually used as a motif to modify siRNA-nanodelivery vectors to improve tumor-targeting and penetration. However, most of the modifications are realized by covalent conjugation, which normally requires complex preparation processes possibly with low conjugation efficiency and yield, and might lower its bioactivity. To avoid this, here, we presented an alternative physical method to decorate iRGD on nanopolymersomes via facile self-assembly in water. siVEGF was chosen as a siRNA model, and lipopolysaccharide-amine nanopolymersomes (NPs), an efficient cytosolic delivery vector developed by our group, was used as an original vector. By successively incubating siVEGF with NPs, followed by adding iRGD, a siVEGF-loaded NPs functionalized with iRGD (siRNA/iRGD-NPs) was obtained. The properties of iRGD-NPs or siRNA/iRGD-NPs were evaluated in vitro and in vivo. iRGD is efficiently introduced onto NPs with different amounts, which can be precisely controlled by the feeding ratio. The introduced iRGD keeps tumor-targeting and -penetrating bioactivity, which endows iRGD-NPs with ~100% of tumor-cell uptake and excellent tumor spheroid-penetration, and thus iRGD-NPs can efficiently deliver siVEGF to significantly inhibit angiogenesis in zebrafish and tumor growth in nude mice bearing breast cancer without obvious toxicity. This study provides a facile physical method to decorate nanodelivery vectors with iRGD for effective targeted siRNA anti-tumor therapy.

Immunogenicity and protective efficacy of a prototype pneumococcal bioconjugate vaccine

Capsular polysaccharides (CPSs), with which most pathogenic bacterial surfaces are decorated, have been used as the main components of glycoconjugate vaccines against bacterial diseases in clinical practice worldwide. Pneumococcal conjugate vaccines (PCVs) are administered globally to prevent invasive pneumococcal disease (IPD). While PCVs have played important roles in controlling IPD in all age groups, their empirical, and labor-intensive chemical conjugation yield poorly characterized, heterogeneous, and variably immunogenic vaccines, with poor immune responses in high-risk populations such as the elderly and patients with weak immune systems. We previously developed a method that bypasses the dependency of chemical conjugation and instead exploits prokaryotic glycosylation systems to produce pneumococcal conjugate vaccines. The bioconjugation platform relies on a conjugating enzyme to transfer a bacterial polysaccharide to an engineered carrier protein all within the lab safe bacterium E. coli. In these studies, we demonstrate that a serotype 8 pneumococcal bioconjugate vaccine is highly immunogenic and elicits functionally protective anti-serotype 8 antibody responses. Specifically, using multiple models we show that mice immunized with multiple doses of a serotype 8 bioconjugate vaccine elicit antibody responses that mediate opsonophagocytic killing, protect mice from systemic infection, and decrease the ability of serotype 8 pneumococci to colonize the nasopharynx and disseminate. Collectively, these studies demonstrate the utility of bioconjugation to produce efficacious pneumococcal conjugate vaccines.

Thiol–Norbornene Photoclick Chemistry for Grafting Antimicrobial Peptides onto Chitosan to Create Antibacterial Biomaterials

Covalent conjugation of antimicrobial peptides (AMP) is considered an effective approach to overcome their potential degradation in vivo. As the high cost of AMP-based therapies is one of the major hurdles toward their clinical application, it is urgently needed to explore high yield reactions. Herein, the highly efficient thiol–norbornene photoclick chemistry (TNPC) was explored for AMP grafting onto chitosan. Norbornenes were introduced onto chitosan (NorChit) in an aqueous/organic system, followed by UV-triggered conjugation of N- and C-terminus cysteine-modified Dhvar5 (NorChit-Dhvar5). Up to 0.38 norbornene groups per chitosan repeating unit and up to 43% conjugation yield in NorChit-Nt-Dhvar5 (80 μmol of Dhvar5/g) were achieved, while in NorChit-Ct-Dhvar5 conjugation yield was 30% (55 μmol of Dhvar5/g). Finally, NorChit-Dhvar5 ultrathin films showed up to a 35% reduction of total adhered Gram-positive Staphylococcus epidermidis and increased the adhesion and killing of Gram-negative Pseudomonas aeruginosa compared to unmodified chitosan. Moreover, NorChit-Dhvar5 was noncytotoxic to human neonatal dermal fibroblasts, according to ISO 10993-1. Overall, our findings indicate TNPC as a high yield strategy for AMP grafting onto norbornene-functionalized biopolymers toward the fabrication of antibacterial biomaterials.

Conjugation of soy protein isolate (SPI) with pectin: effects of structural modification of the grafting polysaccharide

Recently, there has been a great interest in enhancing the emulsifying properties of soy protein isolate (SPI) by Maillard reaction. As a commonly-used grafting polysaccharide, pectin has proved useful in modifying proteins. However, effects of its structural characteristics on conjugation are still not fully understood. To address this problem, we employed alkaline or/and enzymatic treatments to modify pectin and obtained three modified samples. Structural characteristics of pectin, including the molecular weight, degree of methoxylation and acetylation, and monosaccharide compositions were measured. When conjugated with SPI, pectin with lower molecular weight and less main chains induced higher conjugate yield. Fluorescence intensity and surface hydrophobicity of all conjugates markedly reduced compared to the original SPI, suggesting a more loosened protein structure after Maillard reaction. In this study, the enzymolysis pectin proved an optimum grafting polysaccharide considering the simple preparation procedures and the highest emulsifying properties of its resulting conjugates.

Linear Polyglycerol for N-terminal-selective Modification of Interleukin-4

Polymer conjugation to biologics is of key interest to the pharmaceutical industry for the development of potent and long acting biotherapeutics, with poly(ethylene glycol) (PEG) being the gold standard. Within the last years, unwanted PEG-related side effects (immunological reactions, antibody formation) arose, therefore creating several attempts to establish alternative polymers with similar potential to PEG. In this article, we synthesized N-terminal bioconjugates of the potential therapeutic human interleukin-4 (hIL-4 WT) with linear polyglycerol (LPG) of 10 and 40 kDa and compared it with its PEG analogs of same nominal weights. Polyglycerol is a highly hydrophilic polymer with good biocompatibility and therefore represents an alternative polymer to PEG.Both polymer types resulted in similar conjugation yields, comparable hydrodynamic sizes and an unaltered secondary structure of the protein after modification. LPG- and PEG-bioconjugates remained stable in human plasma, whereas binding to human serum albumin (HSA) decreased after polymer modification. Furthermore, only minor differences in bioactivity were observed between LPG- and PEG-bioconjugates of same nominal weights. The presented findings are promising for future pharmacokinetic evaluation of hIL-4-polymer bioconjugates.

Thermostable and Long-Circulating Albumin-Conjugated Arthrobacter globiformis Urate Oxidase.

Urate oxidase derived from Aspergillus flavus has been investigated as a treatment for tumor lysis syndrome, hyperuricemia, and gout. However, its long-term use is limited owing to potential immunogenicity, low thermostability, and short circulation time in vivo. Recently, urate oxidase isolated from Arthrobacter globiformis (AgUox) has been reported to be thermostable and less immunogenic than the Aspergillus-derived urate oxidase. Conjugation of human serum albumin (HSA) to therapeutic proteins has become a promising strategy to prolong circulation time in vivo. To develop a thermostable and long-circulating urate oxidase, we investigated the site-specific conjugation of HSA to AgUox based on site-specific incorporation of a clickable non-natural amino acid (frTet) and an inverse electron demand Diels–Alder reaction. We selected 14 sites for frTet incorporation using the ROSETTA design, a computational stability prediction program, among which AgUox containing frTet at position 196 (Ag12) exhibited enzymatic activity and thermostability comparable to those of wild-type AgUox. Furthermore, Ag12 exhibited a high HSA conjugation yield without compromising the enzymatic activity, generating well-defined HSA-conjugated AgUox (Ag12-HSA). In mice, the serum half-life of Ag12-HSA was approximately 29 h, which was roughly 17-fold longer than that of wild-type AgUox. Altogether, this novel formulated AgUox may hold enhanced therapeutic efficacy for several diseases.

Protein/polysaccharide conjugation via Maillard reactions in an aqueous media: Impact of protein type, reaction time and temperature

It is generally accepted that protein-polysaccharide interactions improve the functional properties of proteins. One of the main issues, to our knowledge, of the effects of protein type and reaction conditions, is a lack of production of Maillard conjugates. This paper focuses on the effects of protein type and ratio, reaction time and temperature on the protein/polysaccharide conjugation by Maillard reactions in an aqueous media. Response Surface Methodology D-Optimal Design was used to optimize the results. Protein type, protein-polysaccharide ratio, reaction time (min) and temperature (°C) were described as factors, whereas conjugation yield (CY), conjugation efficiency (CE), and protein solubility (PS) were described as responses. The optimized parameters for maximum CE and CY determined were found to be as; reaction temperature, 90 °C; reaction time, 60 min; protein type, faba bean protein concentrate (FPC), and protein-polysaccharide ratio 1:2. SEM images, FT-IR spectra, amino acid profile, SDS-PAGE and browning index were the analyses conducted for the optimum point (FPC-Carrageenan). Conjugation was achieved successfully by the confirmation of FT-IR results. Browning index and SDS-PAGE results revealed that Maillard reactions were limited in the initial stage of the Schiff base. These findings confirm that FPC was the most suitable protein type for preparing Maillard-glycated conjugates as a promising agent.

Construction and evaluation of ovalbumin-pullulan nanogels as a potential delivery carrier for curcumin

Preparation of protein/polysaccharide nanocomplexes for delivering environment-sensitive bioactive compounds is significant in the fields of functional foods and pharmaceuticals. In this work, ovalbumin-pullulan (OVA-Pul) nanogels were fabricated through Maillard reaction combined with heat treatment. The results of SDS-PAGE, circular dichroism and conjugation yield (84.96%) confirmed the covalent crosslinking of ovalbumin to pullulan. Dynamic light scattering measurements indicated that nanogels and curcumin-loaded nanogels exhibited small particle diameter at around 190 nm and 160 nm, and excellent polydispersity index at 0.227 and 0.146, respectively. OVA-Pul nanogels showed good encapsulation efficiency (88.38%) and loading capacity (8.78%) for curcumin. Transmission electron microscope observations and in vitro gastrointestinal digestion suggested that OVA-Pul nanogels facilitated the controlled release of curcumin and the spherical structure of curcumin-loaded nanogels was damaged during digestion. Notably, both nanogels and curcumin-loaded nanogels showed desirable storage stability during 30 d. Hence, OVA-Pul nanogels have the potential for effectively delivering nutrients and drugs.

Multivalent Albumin-Neonatal Fc Receptor Interactions Mediate a Prominent Extension of the Serum Half-Life of a Therapeutic Protein.

Human serum albumin (HSA) has been used to extend the serum half-life of therapeutic proteins owing to its exceptionally long serum half-life via the neonatal Fc receptor (FcRn)-mediated recycling mechanism. In most cases, only one HSA molecule was conjugated to a therapeutic protein, leading to a limited extension of the serum half-life. In this study, we hypothesized that conjugation of multiple HSA molecules to a therapeutic protein significantly further extends the serum half-life via multivalent HSA-FcRn interactions. We chose urate oxidase (Uox), a tetrameric therapeutic protein used for the treatment of gout, as a model. In previous studies, only one HSA molecule was site-specifically conjugated to one Uox because of poor conjugation yield of the relatively slow bio-orthogonal chemistry, strain-promoted azide-alkyne cycloaddition (SPAAC). To increase the number of HSA molecules conjugated to one Uox, we employed the faster bio-orthogonal chemistry, inverse electron demand Diels-Alder reaction (IEDDA). We site-specifically introduced the phenylalanine analog with a fast-reacting tetrazine group (frTet) into position 174 of each subunit of Uox. We then achieved site-specific HSA conjugation to each subunit of Uox via IEDDA, generating Uox conjugated to four HSA molecules (Uox-HSA4), with a small portion of Uox conjugated to three HSA molecules (Uox-HSA3). We characterized Uox-HSA4 as well as Uox variants conjugated to one or two HSA molecules prepared via SPAAC (Uox-HSA1 or Uox-HSA2). The enzyme activity of all three Uox-HSA conjugates was comparable to that of unmodified Uox. We found out that an increase in HSA molecules conjugated to Uox (multiple albumin-conjugated therapeutic protein) enhanced FcRn binding and consequently prolonged the serum half-life in vivo. In particular, the conjugation of four HSA molecules to Uox led to a prominent extension of the serum half-life (over 21 h), which is about 16-fold longer than that of Uox-WT.