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Therapeutic proteins, pivotal for treating diverse human diseases due to their biocompatibility and high selectivity, often face challenges such as rapid serum clearance, enzymatic degradation, and immune responses. To address these issues and enable prolonged therapeutic efficacy, techniques to extend the serum half-life of therapeutic proteins are crucial. The AlbuCatcher, a conjugate of human serum albumin (HSA) and SpyCatcher, was proposed as a general technique to extend the serum half-life of diverse therapeutic proteins. HSA, the most abundant blood protein, exhibits a long intrinsic half-life through Fc receptor (FcRn)-mediated recycling. The SpyTag/SpyCatcher (ST/SC) system, known for forming irreversible isopeptide bonds, was employed to conjugate HSA and therapeutic proteins. Site-specific HSA conjugation to SC was achieved using an inverse electron-demand Diels-Alder (IEDDA) reaction, minimizing activity loss. Using urate oxidase (Uox) as a model protein with a short half-life, the small ST was fused to generate Uox-ST. Then, HSA-conjugated Uox (Uox-HSA) was successfully prepared via the Uox-ST/AlbuCatcher reaction. In vitro enzyme assays demonstrated that the impact of ST fusion and HSA conjugation on Uox enzymatic activity is negligible. Pharmacokinetics studies in mice revealed that Uox-HSA exhibits a significantly longer serum half-life (about 18 h) compared to Uox-WT (about 2 h). This extended half-life is attributed to FcRn-mediated recycling of HSA-conjugated Uox, demonstrating the effectiveness of the AlbuCatcher strategy in enhancing the pharmacokinetics of therapeutic proteins.
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Rare earth elements (REEs), including those in the lanthanide series, are crucial components essential for clean energy transitions, but they originate from geographically limited regions. Exploiting new and diverse supply sources is vital to facilitating a clean energy future. Hence, we explored the recovery of REEs from coal fly ash (FA), a complex, low-grade industrial feedstock that is currently underutilized (leachate concentrations of REEs in FA are < 0.003 mol%). Herein, we demonstrated the thermo-responsive genetically encoded REE-selective elastin-like polypeptides (RELPs) as a recyclable bioengineered protein adsorbent for the selective retrieval of REEs from coal fly ash over multiple cycles. The results showed that RELPs could be efficiently separated using temperature cycling and reused with high stability, as they retained â¼95% of their initial REE binding capacity even after four cycles. Moreover, RELPs selectively recovered high-purity REEs from the simulated solution containing one representative REE in the range of 0.0001-0.005 mol%, resulting in up to a 100,000-fold increase in REE purity. This study offers a sustainable approach to diversifying REE supplies by recovering REEs from low-grade coal fly ash in industrial wastes and provides a scientific basis for the extraction of high-purity REEs for industrial purposes.
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BACKGROUND: The use of single-chain variable fragments (scFvs) for treating human diseases, such as cancer and immune system disorders, has attracted significant attention. However, a critical drawback of scFv is its extremely short serum half-life, which limits its therapeutic potential. Thus, there is a critical need to prolong the serum half-life of the scFv for clinical applications. One promising serum half-life extender for therapeutic proteins is human serum albumin (HSA), which is the most abundant protein in human serum, known to have an exceptionally long serum half-life. However, conjugating a macromolecular half-life extender to a small protein, such as scFv, often results in a significant loss of its critical properties. RESULTS: In this study, we conjugated the HSA to a permissive site of scFv to improve pharmacokinetic profiles. To ensure minimal damage to the antigen-binding capacity of scFv upon HSA conjugation, we employed a site-specific conjugation approach using a heterobifunctional crosslinker that facilitates thiol-maleimide reaction and inverse electron-demand Diels-Alder reaction (IEDDA). As a model protein, we selected 4D5scFv, derived from trastuzumab, a therapeutic antibody used in human epithermal growth factor 2 (HER2)-positive breast cancer treatment. We introduced a phenylalanine analog containing a very reactive tetrazine group (frTet) at conjugation site candidates predicted by computational methods. Using the linker TCO-PEG4-MAL, a single HSA molecule was site-specifically conjugated to the 4D5scFv (4D5scFv-HSA). The 4D5scFv-HSA conjugate exhibited HER2 binding affinity comparable to that of unmodified 4D5scFv. Furthermore, in pharmacokinetic profile in mice, the serum half-life of 4D5scFv-HSA was approximately 12 h, which is 85 times longer than that of 4D5scFv. CONCLUSIONS: The antigen binding results and pharmacokinetic profile of 4D5scFv-HSA demonstrate that the site-specifically albumin-conjugated scFv retained its binding affinity with a prolonged serum half-life. In conclusion, we developed an effective strategy to prepare site-specifically albumin-conjugated 4D5scFv, which can have versatile clinical applications with improved efficacy.
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Achieving efficient and site-specific conjugation of therapeutic protein to polymer is crucial to augment their applicability in the realms of biomedicine by improving their stability and enzymatic activity. In this study, we exploited tetrazine bioorthogonal chemistry to achieve the site-specific conjugation of bottlebrush polymers to urate oxidase (UOX), a therapeutic protein for gout treatment. An azido-functionalized zwitterionic bottlebrush polymer (N3-ZBP) using a "grafting-from" strategy involving RAFT and ATRP methods was synthesized, and a trans-cyclooctene (TCO) moiety was introduced at the polymer end through the strain-promoted azide-alkyne click (SPAAC) reaction. The subsequent coupling between TCO-incorporated bottlebrush polymer and tetrazine-labeled UOX using a fast and safe bioorthogonal reaction, inverse electron demand Diels-Alder (IEDDA), led to the formation of UOX-ZBP conjugates with a 52% yield. Importantly, the enzymatic activity of UOX remained unaffected following polymer conjugation, suggesting a minimal change in the folded structure of UOX. Moreover, UOX-ZBP conjugates exhibited enhanced proteolytic resistance and reduced antibody binding, compared to UOX-wild type. Overall, the present findings reveal an efficient and straightforward route for synthesizing protein-bottlebrush polymer conjugates without compromising the enzymatic activity while substantially reducing proteolytic degradation and antibody binding.
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Química Clic , Reacción de Cicloadición , Polímeros , Urato Oxidasa , Urato Oxidasa/química , Química Clic/métodos , Polímeros/química , Ciclooctanos/química , Humanos , Azidas/química , Alquinos/químicaRESUMEN
Genome-scale metabolic model (GEM) can be used to simulate cellular metabolic phenotypes under various environmental or genetic conditions. This study utilized the GEM to observe the internal metabolic fluxes of recombinant Escherichia coli producing gamma-aminobutyric acid (GABA). Recombinant E. coli was cultivated in a fermenter under three conditions: pH 7, pH 5, and additional succinic acids. External fluxes were calculated from cultivation results, and internal fluxes were calculated through flux optimization. Based on the internal flux analysis, glycolysis and pentose phosphate pathways were repressed under cultivation at pH 5, even though glutamate dehydrogenase increased GABA production. Notably, this repression was halted by adding succinic acid. Furthermore, proper sucA repression is a promising target for developing strains more capable of producing GABA.
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Escherichia coli , Ácido gamma-Aminobutírico , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/biosíntesis , Concentración de Iones de Hidrógeno , Fermentación , Glucólisis , Ácido Succínico/metabolismo , Vía de Pentosa Fosfato , Análisis de Flujos Metabólicos , Modelos Biológicos , Reactores Biológicos/microbiología , Glutamato Deshidrogenasa/metabolismo , Glutamato Deshidrogenasa/genética , Ingeniería Metabólica/métodosRESUMEN
The pressing challenge of cancer's high mortality and invasiveness demands improved therapeutic approaches. Targeting the nutrient dependencies within cancer cells has emerged as a promising approach. This study is dedicated to demonstrating the potential of arginine depletion for cancer treatment. Notably, the focus centers on arginine decarboxylase (RDC), a pH-dependent enzyme expecting enhanced activity within the slightly acidic microenvironments of tumors. To investigate the effect of a single-site mutation on the catalytic efficacy of RDC, diverse amino acids, including glycine, alanine, phenylalanine, tyrosine, tryptophan, p-azido-phenylalanine, and a phenylalanine analog with a hydrogen-substituted tetrazine, were introduced at the crucial threonine site (position 39) in the multimer-forming interface. Remarkably, the introduction of either a natural or a non-natural aromatic amino acid at position 39 substantially boosted enzymatic activity, while amino acids with smaller side chains did not show the same effect. This enhanced enzymatic activity is likely attributed to the reinforced formation of multimer structures through favorable interactions between the introduced aromatic amino acid and the neighboring subunit. Noteworthy, at slightly acidic pH, the RDC variant featuring tryptophan at position 39 demonstrated augmented cytotoxicity against tumor cells compared to the wild-type RDC. This attribute aligns with the tumor microenvironment and positions these variants as potential candidates for targeted cancer therapy.
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Aminoácidos Aromáticos , Carboxiliasas , Triptófano , Triptófano/química , Aminoácidos/metabolismo , Tirosina , Fenilalanina , ArgininaRESUMEN
BACKGROUND: Exogenously providing engineered Uox with enhanced half-life is one of the important urate-lowering treatments for gout. The potential of PAT101, a recombinant human albumin (rHA)-conjugated variant, was evaluated and compared as a novel gout treatment through various in vivo studies with PAT101 and competing drugs. METHODS: PAT101 was produced by site-specific conjugation of rHA and Aspergillus flavus Uox (AfUox-rHA) through clickable non-natural amino acid (frTet) and Inverse electron demand Diels-Alder (IEDDA) reaction. In vivo pharmacokinetics, efficacy tests and in vitro immunogenetic assay were performed after single or multiple doses of PAT101 and its competitors in BALB/c mice, transgenic (TG) mice, Sprague-Dawley (SD) rats, and non-human primate (NHP). RESULTS: The half-life of PAT101 in single-dose treated TG mice was more than doubled compared to pegloticase. In SD rats with 4 weeks of repeated administration of rasburicase, only 24% of Uox activity remained, whereas in PAT101, it was maintained by 86%. In the Uox KO model, the survival rate of PAT101 was comparable to that of pegloticase. In addition, human PBMC-based CD4+/CD8+ T-cell activation analysis demonstrated that PAT101 has a lower immune response compared to the original drug, rasburicase. CONCLUSION: All results suggest that this rHA-conjugated AfUox, PAT101, can be provided as a reliable source of Uox for gout treatment.
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Gota , Urato Oxidasa , Ratones , Animales , Ratas , Humanos , Urato Oxidasa/uso terapéutico , Leucocitos Mononucleares/metabolismo , Ratas Sprague-Dawley , Gota/tratamiento farmacológico , Supresores de la Gota/uso terapéutico , Ratones Transgénicos , Polietilenglicoles/uso terapéutico , Albúminas/uso terapéuticoRESUMEN
It is challenging to capture carbon dioxide (CO2), a major greenhouse gas in the atmosphere, due to its high chemical stability. One potential practical solution to eliminate CO2 is to convert CO2 into formate using hydrogen (H2) (CO2 hydrogenation), which can be accomplished with inexpensive hydrogen from sustainable sources. While industrial flue gas could provide an adequate source of hydrogen, a suitable catalyst is needed that can tolerate other gas components, such as carbon monoxide (CO) and oxygen (O2), potential inhibitors. Our proposed CO2 hydrogenation system uses the hydrogenase derived from Ralstonia eutropha H16 (ReSH) and formate dehydrogenase derived from Methylobacterium extorquens AM1 (MeFDH1). Both enzymes are tolerant to CO and O2, which are typical inhibitors of metalloenzymes found in flue gas. We have successfully demonstrated that combining ReSH- and MeFDH1-immobilized resins can convert H2 and CO2 in real flue gas to formate via a nicotinamide adenine dinucleotide-dependent cascade reaction. We anticipated that this enzyme system would enable the utilization of diverse H2 and CO2 sources, including waste gases, biomass, and gasified plastics.
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Single-chain variable fragments (scFvs) have been recognized as promising agents in cancer therapy. However, short serum half-life of scFvs often limits clinical application. Fusion to albumin affibody (ABD) is an effective and convenient half-life extension strategy. Although one terminus of scFv is available for fusion of ABD, it is also frequently used for fusion of useful moieties such as small functional proteins, cytokines, or antibodies. Herein, we investigated the internal linker region for ABD fusion instead of terminal region, which was rarely explored before. We constructed two internally ABD-inserted anti-HER2 4D5scFv (4D5-ABD) variants, which have short (4D5-S-ABD) and long (4D5-L-ABD) linker length respectively. The model structures of these 4D5scFv and 4D5-ABD variants predicted using the deep learning-based protein structure prediction program (AlphaFold2) revealed high similarity to either the original 4D5scFv or the ABD structure, implying that the functionality would be retained. Designed 4D5-ABD variants were expressed in the bacterial expression system and characterized. Both 4D5-ABD variants showed anti-HER2 binding affinity comparable with 4D5scFv. Binding affinity of both 4D5-ABD variants against albumin was also comparable. In a pharmacokinetic study in mice, the 4D5-ABD variants showed a significantly prolonged half-life of 34 h, 114 times longer than that of 4D5scFv. In conclusion, we have developed a versatile scFv platform with enhanced pharmacokinetic profiles with an aid of deep learning-based structure prediction.
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Hydrogen gas obtained from cheap or sustainable sources has been investigated as an alternative to fossil fuels. By using hydrogenase (H2ase) and formate dehydrogenase (FDH), H2 and CO2 gases can be converted to formate, which can be conveniently stored and transported. However, developing an enzymatic process that converts H2 and CO2 obtained from cheap sources into formate is challenging because even a very small amount of O2 included in the cheap sources damages most H2ases and FDHs. In order to overcome this limitation, we investigated a pair of oxygen-tolerant H2ase and FDH. We achieved the cascade reaction between H2ase from Ralstonia eutropha H16 (ReSH) and FDH from Rhodobacter capsulatus (RcFDH) to convert H2 and CO2 to formate using in situ regeneration of NAD+/NADH in the presence of O2.
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3-arylpropiolonitriles (APN) are promising alternatives to maleimide for chemo-selective thiol conjugation, because the reaction product has a remarkably hydrolytic stability compared with that of thiol-maleimide reactions in vitro. However, whether cysteine modification with APN enhances stability in vivo compared to thiol-maleimide reactions remains unclear, probably due to the too short in vivo serum half-life of a protein to observe significant cleavage of thiol-maleimide/-APN reaction products. The conjugation of human serum albumin (HSA) to a therapeutic protein reportedly prolongs the in vivo serum half-life. To evaluate the in vivo stability of the thiol-APN reaction product, we prepared HSA-conjugated Arthrobacter globiformis urate oxidase (AgUox), a therapeutic protein for gout treatment. Site-specific HSA conjugation to AgUox was achieved by combining site-specific incorporation of tetrazine containing an amino acid (frTet) into AgUox and a crosslinker containing trans-cyclooctene and either thiol-maleimide (AgUox-MAL-HSA) or -APN chemistry (AgUox-APN-HSA). Substantial cleavage of the thioester of AgUox-MAL-HSA was observed in vitro, whereas no cleavage of the thiol-APN product of AgUox-APN-HSA was observed. Furthermore, the in vivo serum half-life of AgUox-APN-HSA in the late phase was significantly longer than that of AgUox-MAL-HSA. Overall, these results demonstrate that the thiol-APN chemistry enhanced the in vivo stability of the HSA-conjugated therapeutic protein.
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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.
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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.
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Excipientes/química , Antígenos de Histocompatibilidad Clase I/química , Receptores Fc/química , Albúmina Sérica Humana/química , Urato Oxidasa/farmacocinética , Animales , Reacción de Cicloadición , Pruebas de Enzimas , Femenino , Semivida , Inyecciones Intravenosas , Ratones , Urato Oxidasa/administración & dosificación , Urato Oxidasa/químicaRESUMEN
Glucagon-like peptide-1 (GLP-1) is a peptide hormone with tremendous therapeutic potential for treating type 2 diabetes mellitus. However, the short half-life of its native form is a significant drawback. We previously prolonged the plasma half-life of GLP-1 via site-specific conjugation of human serum albumin (HSA) at position 16 of recombinant GLP-1 using site-specific incorporation of p-azido-phenylalanine (AzF) and strain-promoted azide-alkyne cycloaddition (SPAAC). However, the resulting conjugate GLP1_8G16AzF-HSA showed only moderate in vivo glucose-lowering activity, probably due to perturbed interactions with GLP-1 receptor (GLP-1R) caused by the albumin-linker. To identify albumin-conjugated GLP-1 variants with enhanced in vivo glucose-lowering activity, we investigated the conjugation of HSA to a C-terminal region of GLP-1 to reduce steric hindrance by the albumin-linker using two different conjugation chemistries. GLP-1 variants GLP1_8G37AzF-HSA and GLP1_8G37C-HSA were prepared using SPAAC and Michael addition, respectively. GLP1_8G37C-HSA exhibited a higher glucose-lowering activity in vivo than GLP1_8G16AzF-HSA, while GLP1_8G37AzF-HSA did not. Another GLP-1 variant, GLP1_8A37C-HSA, had a glycine to alanine mutation at position 8 and albumin at its C-terminus and exhibited in vivo glucose-lowering activity comparable to that of GLP1_8G37C-HSA, despite a moderately shorter plasma half-life. These results showed that site-specific HSA conjugation to the C-terminus of GLP-1 via Michael addition could be used to generate GLP-1 variants with enhanced glucose-lowering activity and prolonged plasma half-life in vivo.
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An inverse-electron-demand Diels-Alder (IEDDA) reaction using genetically encoded tetrazine variants enables rapid bioconjugation for diverse applications in vitro and in cellulo. However, in vivo bioconjugation using genetically encoded tetrazine variants is challenging, because the IEDDA coupling reaction competes with rapid elimination of reaction partners in vivo. Here, we tested the hypothesis that a genetically encoded phenylalanine analogue containing a hydrogen-substituted tetrazine (frTet) would increase the IEDDA reaction rate, thereby allowing for successful bioconjugation in vivo. We found that the in vitro IEDDA reaction rate of superfolder green fluorescent protein (sfGFP) containing frTet (sfGFP-frTet) was 12-fold greater than that of sfGFP containing methyl-substituted tetrazine (sfGFP-Tet_v2.0). Additionally, sfGFP variants encapsulated with chitosan-modified, pluronic-based nanocarriers were delivered into nude mice or tumor-bearing mice for in vivo imaging. The in vivo-delivered sfGFP-frTet exhibited almost complete fluorescence recovery upon addition of trans-cyclooctene via the IEDDA reaction within 2 h, whereas sfGFP-Tet_v2.0 did not show substantial fluorescence recovery. These results demonstrated that the genetically encoded frTet allows an almost complete IEDDA reaction in vivo upon addition of trans-cyclooctene, enabling temporal control of in vivo bioconjugation in a very high yield.
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Reacción de Cicloadición/métodos , Ciclooctanos/química , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/química , Compuestos Heterocíclicos con 1 Anillo/química , Fenilalanina/análogos & derivados , Animales , Electrones , Proteínas Fluorescentes Verdes/genética , Masculino , Ratones , Ratones Desnudos , Fenilalanina/genéticaRESUMEN
Conjugation of serum albumin or one of its ligands (such as fatty acid) has been an effective strategy to prolong the serum half-lives of drugs via neonatal Fc receptor (FcRn)-mediated recycling of albumin. So far, fatty acid (FA) has been effective in prolonging the serum half-lives for therapeutic peptides and small proteins, but not for large therapeutic proteins. Very recently, it was reported a large protein conjugated to FA competes with the binding of FcRn with serum albumin, leading to limited serum half-life extension, because primary FA binding sites in serum albumin partially overlap with FcRn binding sites. In order to prevent such competition, longer linkers between FA and the large proteins were required. Herein, we hypothesized that small proteins do not cause substantial competition for FcRn binding to albumin, resulting in the extended serum half-life. Using a small protein (28 kDa), we investigated whether the intramolecular distance in FA-protein conjugate affects the FcRn binding with albumin and serum half-life using linkers with varying lengths. Unlike with the FA-conjugated large protein, all FA-conjugated small proteins with different linkers exhibited comparable the FcRn binding to albumin and extended serum half-life.
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Therapeutic proteins are attractive candidates for the treatment of human diseases. However, their short half-life often limits their clinical application. To overcome this problem, injectable hydrogels have been developed as depots for controlled release of therapeutic proteins, but these systems have not yet achieved the desired extended, sustained drug release profile. Our strategy herein was to implement selective and strong interactions between the hydrogels and therapeutic proteins. Specifically, we investigated whether strong and specific interactions between human serum albumin (HSA) and albumin-binding peptide (ABP) can be used to achieve extended release of urate oxidase (Uox), a therapeutic protein for hyperuricemia treatment, from pH- and temperature-sensitive injectable hydrogels consisting of poly(ethylene glycol)-poly(ß-amino ester urethane) (PEG-PAEU) copolymer. Thus, HSA was conjugated to Uox (Uox-HSA) and ABP was introduced in PEG-PAEU (PEG-PAEU-ABP). Polymers, conjugates, and hydrogels were extensively characterized for their physicochemical characteristics and in vivo efficacy in a hyperuricemia mouse model. Briefly, the hydrogels exhibited good injectability, in vitro biocompatibility and extended drug release, and in vivo gel formation and degradability. The serum half-life of the Uox-HSA loaded in PEG-PAEU-ABP hydrogels was ~96 h in mice, which was ~88, ~5.5, and ~2 times longer than that of free native Uox, free Uox-HSA, and Uox-HSA loaded in PEG-PAEU hydrogels, respectively. In the hyperuricemia mouse model, Uox-HSA loaded in PEG-PAEU-ABP hydrogels exhibited a substantially extended period of uric acid-lowering efficacy. These results clearly show that by applying ABP-HSA strong interaction to injectable hydrogels and therapeutic protein, the concentration of the therapeutic protein can be maintained for a long period in vivo, prolonging its therapeutic effect. Further, our approach can be tailored to accommodate other therapeutic proteins, which potentially expands the clinical applicability range of these systems.
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Hiperuricemia , Urato Oxidasa , Animales , Preparaciones de Acción Retardada , Sistemas de Liberación de Medicamentos , Liberación de Fármacos , Hidrogeles , Hiperuricemia/tratamiento farmacológico , Ratones , Polietilenglicoles , Albúmina Sérica HumanaRESUMEN
The number of therapeutic peptides for human treatment is growing rapidly. However, their development faces two major issues: the poor yield of large peptides from conventional solid-phase synthesis, and the intrinsically short serum half-life of peptides. To address these issues, we investigated a platform for the production of a recombinant therapeutic peptide with an extended serum half-life involving the site-specific conjugation of human serum albumin (HSA). HSA has an exceptionally long serum half-life and can be used to extend the serum half-lives of therapeutic proteins and peptides. We used glucagon-like-peptide 1 (GLP-1) as a model peptide in the present study. A "clickable" non-natural amino acid-p-azido-l-phenylalanine (AzF)-was incorporated into three specific sites (V16, Y19, and F28) of a GLP-1 variant, followed by conjugation with HSA through strain-promoted azide-alkyne cycloaddition. All three HSA-conjugated GLP-1 variants (GLP1_16HSA, GLP1_19HSA, and GLP1_28HSA) exhibited comparable serum half-lives in vivo. However, the three GLP1_HSA variants had different in vitro biological activities and in vivo glucose-lowering effects, demonstrating the importance of site-specific HSA conjugation. The platform described herein could be used to develop other therapeutic peptides with extended serum half-lives.
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Mussel adhesive proteins (MAPs) have great potential as bioglues, particularly in wet conditions. Although in vivo residue-specific incorporation of 3,4-dihydroxyphenylalanine (Dopa) in tyrosine-auxotrophic Escherichia coli cells allows for production of Dopa-incorporated bioengineered MAPs (dMAPs), the low production yield hinders the practical application of dMAPs. This low production yield of dMAPs is due to low translational activity of a noncanonical amino acid, Dopa, in E. coli cells. Herein, to enhance the production yield of dMAPs, we investigated the coexpression of Dopa-recognizing tyrosyl-tRNA synthetases (TyrRSs). To use the Dopa-specific Methanococcus jannaschii TyrRS (MjTyrRS-Dopa), we altered the anticodon of tyrosyl-tRNA amber suppressor into AUA (MjtRNATyrAUA ) to recognize a tyrosine codon (AUA). Co-overexpression of MjTyrRS-Dopa and MjtRNATyrAUA increased the production yield of Dopa-incorporated MAP foot protein type 3 (dfp-3) by 57%. Similarly, overexpression of E. coli TyrRS (EcTyrRS) led to a 72% higher production yield of dfp-3. Even with coexpression of Dopa-recognizing TyrRSs, dfp-3 has a high Dopa incorporation yield (over 90%) compared to ones prepared without TyrRS coexpression.
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Dihidroxifenilalanina/genética , Moluscos/genética , Ingeniería de Proteínas/métodos , Proteínas/genética , Animales , Codón , Escherichia coli/genética , Methanocaldococcus/genética , Biosíntesis de ProteínasRESUMEN
Therapeutic proteins are indispensable for treatment of various human diseases. However, intrinsic short serum half-lives of proteins are still big hurdles for developing new therapeutic proteins or expanding applications of existing ones. Urate oxidase (Uox) is a therapeutic protein clinically used for treatment of hyperuricemia. Due to its short half-life, its application for gout treatment requires prolonging the half-life in vivo. Conjugation of a fatty acid (FA), a serum albumin (SA) ligand, to therapeutic proteins/peptides is an emerging strategy to prolong serum half-life presumably via neonatal Fc receptor (FcRn)-mediated recycling. FA conjugation was proven effective for peptides and small proteins (less than 28 kDa), but not for Uox (140 kDa). We hypothesized that the intramolecular distance in the conjugate of FA and Uox is a critical factor for effective FcRn-mediated recycling. In order to control the intramolecular distance in the conjugate, we varied linker lengths between Uox and palmitic acid (PA). There was a linear correlation between the linker length and serum half-life of PA-conjugated Uox (Uox-PA) conjugates. The longer linker led to about 7-fold greater extension of serum half-life of Uox in mice than the unmodified Uox. The trend in serum half-life extension matched well with that in the tertiary structure formation of FcRn/SA/Uox-PA in vitro. These results demonstrate that the intramolecular distance in the conjugate of Uox and FA governs the stable formation of FcRn/SA/FA-conjugated protein and serum half-life extension in vivo. These findings would also contribute to development of effective FAconjugated therapeutic proteins.