Herein,we report the facile conjugation between proteins and water-soluble [60]fullerene derivatives(DC_(60)) under native conditions using SpyTag as a reactive handle.Water-soluble [60] fullerene derivatives were fir...Herein,we report the facile conjugation between proteins and water-soluble [60]fullerene derivatives(DC_(60)) under native conditions using SpyTag as a reactive handle.Water-soluble [60] fullerene derivatives were first prepared via sequential Bingel-Hirsch reaction and "clicked" with SpyTag to give DC_(60)-SpyTag for native conjugation with proteins by the highly efficient SpyTag-SpyCatcher chemistry.The bioconjugation was confirmed by MALDI-TOF MS spectra and SDS-PAGE analysis.The TEM and UVvis spectroscopic study further revealed that the DC_(60) could alter the optical performance and induce aggregation of the target proteins.It thus provides a general and robust method for modifying proteins with C_(60) derivatives and could potentially be adapted for native conjugation between proteins and other nonbiological motifs as well.展开更多
Synthesis of macromolecular systems with precise structural and functional control constitutes a fundamental challenge for materials science and engineering. Development of the ability to construct complex bio-macromo...Synthesis of macromolecular systems with precise structural and functional control constitutes a fundamental challenge for materials science and engineering. Development of the ability to construct complex bio-macromolecular architectures provides a solution to this challenge. The past few years have witnessed the emergence of a new category of peptide-protein chemistry which can covalently stitch together protein]peptide molecules with high specificity under mild physiological conditions. It has thus inspired the concept of genetically encoded click chemistry （GECC）. As a prototype of GECC, SpyTag/ SpyCatcher chemistry has enabled the precise synthesis ofmacromolecules both in vitro and in vivo, exerting precise control over the fundamental properties of these macromolecules including length, sequence, stereochemistry and topology and leading to the creation of diverse biomaterials for a variety of applications. We thus anticipate a potential toolbox of GECC comprising multiple mutually orthogonal, covalent-bond forming peptide-protein reactive pairs with diverse features, which shall bridge synthetic biology and materials science and open up enormous opportunities for biomaterialsin the future.展开更多
Due to the complexity of bioactive ingredients in biological samples,the screening of target proteins is a complex process.Herein,a feasible strategy for directing protein immobilization on silica magnetic beads for l...Due to the complexity of bioactive ingredients in biological samples,the screening of target proteins is a complex process.Herein,a feasible strategy for directing protein immobilization on silica magnetic beads for ligand fishing based on SpyTag/SpyCatcher(ST/SC)-mediated anchoring is presented.Carboxyl functional groups on the surface of silica-coated magnetic beads(SMBs)were coupled with SC using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysulfosuccinimide method,named SC-SMBs.The green fluorescent protein(GFP),as the capturing protein model,was ST-labeled and anchored at a specific orientation onto the surface of SC-SMBs directly from relevant cell lysates via ST/SC self-ligation.The characteristics of the SC-SMBs were studied via electron microscopy,energy dispersive spectroscopy,and Fourier transform infrared spectroscopy.The spontaneity and site-specificity of this unique reaction were confirmed via electrophoresis and fluorescence analyses.Although the alkaline stability of ST-GFP-ligated SC-SMBs was not ideal,the formed isopeptide bond was unbreakable under acidic conditions(0.05 M glycine-HCl buffer,pH 1e6)for 2 h,under 20%ethanol solution within 7 days,and at most temperatures.We,therefore,present a simple and universal strategy for the preparation of diverse protein-functionalized SMBs for ligand fishing,prompting its usage on drug screening and target finding.展开更多
Conjugate vaccines represent one of the most effective means for controlling the occurrence of bacterial diseases.Although nanotechnology has been greatly applied in the field of vaccines,it is seldom used for conjuga...Conjugate vaccines represent one of the most effective means for controlling the occurrence of bacterial diseases.Although nanotechnology has been greatly applied in the field of vaccines,it is seldom used for conjugate vaccine research because it is very difficult to connect polysaccharides and nanocarriers.In this work,an orthogonal and modular biosynthesis method was used to produce nanoconjugate vaccines using the SpyTag/SpyCatcher system.When SpyTag/SpyCatcher system is combined with protein glycosylation technology,bacterial O-polysaccharide obtained from Shigela flexneri 2a can be conjugated onto the surfaces of different virus-like particles(VLPs)in a biocompatible and controlled manner.After confirming the excellent lymph node targeting and humoral immune activation abilities,these nanoconjugate vaccines further induced efficient prophylactic effects against infection in a mouse model.These results demonstrated that natural polysaccharide antigens can be easily connected to VLPs to prepare highly efficient nanoconjugate vaccines.To the best of the researchers1 knowledge,this is the first time VLP-based nanoconjugate vaccines are produced efficiently,and this strategy could be applied to develop various pathogenic nanoconjugate vaccines.展开更多
The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has wreaked havoc around the globe,with no end in sight.The rapid emergence of viral mutants,marked by rapid transmission ...The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has wreaked havoc around the globe,with no end in sight.The rapid emergence of viral mutants,marked by rapid transmission and effective immune evasion,has also posed unprecedented challenges for vaccine development,not least in its speed,mass production,and distribution.Here we report a versatile“plug-and-display”strategy for creating protein vaccines,including those against malaria parasites and SARS-CoV-2,through the combined use of the intrinsically disordered protein ligase SpyStapler and computationally designed viral-like particles.The resulting protein nanoparticles harboring multiple antigens induce potent neutralizing antibody responses in mice,substantially stronger than those induced by the corresponding free antigens.This modular vaccine design enabled by SpyStapler furnishes us with a new weapon for combatting infectious diseases.展开更多
基金the financial support from the National Natural Science Foundation of China(Nos.21925102,21991132 and 21674003)supported by Beijing National Laboratory for Molecular Sciences(No.BNLMS-CXXM-202006)Clinical Medicine Plus X Project of Peking University,Fundamental Research Funds for the Central Universities。
文摘Herein,we report the facile conjugation between proteins and water-soluble [60]fullerene derivatives(DC_(60)) under native conditions using SpyTag as a reactive handle.Water-soluble [60] fullerene derivatives were first prepared via sequential Bingel-Hirsch reaction and "clicked" with SpyTag to give DC_(60)-SpyTag for native conjugation with proteins by the highly efficient SpyTag-SpyCatcher chemistry.The bioconjugation was confirmed by MALDI-TOF MS spectra and SDS-PAGE analysis.The TEM and UVvis spectroscopic study further revealed that the DC_(60) could alter the optical performance and induce aggregation of the target proteins.It thus provides a general and robust method for modifying proteins with C_(60) derivatives and could potentially be adapted for native conjugation between proteins and other nonbiological motifs as well.
基金financial supports from the Research Grants Council of Hong Kong SAR Government to F. Sun (RGC-ECS Nos. #26103915 and Ao E/M-09/12)the 863 Program (No. 2015AA020941)+2 种基金the National Natural Science Foundation of China (Nos. 21474003, 91427304)"1000 Plan (Youth)"the Department of Chemical and Biological Engineering, HKUST for the faculty start-up fund
文摘Synthesis of macromolecular systems with precise structural and functional control constitutes a fundamental challenge for materials science and engineering. Development of the ability to construct complex bio-macromolecular architectures provides a solution to this challenge. The past few years have witnessed the emergence of a new category of peptide-protein chemistry which can covalently stitch together protein]peptide molecules with high specificity under mild physiological conditions. It has thus inspired the concept of genetically encoded click chemistry （GECC）. As a prototype of GECC, SpyTag/ SpyCatcher chemistry has enabled the precise synthesis ofmacromolecules both in vitro and in vivo, exerting precise control over the fundamental properties of these macromolecules including length, sequence, stereochemistry and topology and leading to the creation of diverse biomaterials for a variety of applications. We thus anticipate a potential toolbox of GECC comprising multiple mutually orthogonal, covalent-bond forming peptide-protein reactive pairs with diverse features, which shall bridge synthetic biology and materials science and open up enormous opportunities for biomaterialsin the future.
基金supported by the Zhejiang Foundation Public Welfare Research Project(Authorization No.:LGF19B060006)。
文摘Due to the complexity of bioactive ingredients in biological samples,the screening of target proteins is a complex process.Herein,a feasible strategy for directing protein immobilization on silica magnetic beads for ligand fishing based on SpyTag/SpyCatcher(ST/SC)-mediated anchoring is presented.Carboxyl functional groups on the surface of silica-coated magnetic beads(SMBs)were coupled with SC using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysulfosuccinimide method,named SC-SMBs.The green fluorescent protein(GFP),as the capturing protein model,was ST-labeled and anchored at a specific orientation onto the surface of SC-SMBs directly from relevant cell lysates via ST/SC self-ligation.The characteristics of the SC-SMBs were studied via electron microscopy,energy dispersive spectroscopy,and Fourier transform infrared spectroscopy.The spontaneity and site-specificity of this unique reaction were confirmed via electrophoresis and fluorescence analyses.Although the alkaline stability of ST-GFP-ligated SC-SMBs was not ideal,the formed isopeptide bond was unbreakable under acidic conditions(0.05 M glycine-HCl buffer,pH 1e6)for 2 h,under 20%ethanol solution within 7 days,and at most temperatures.We,therefore,present a simple and universal strategy for the preparation of diverse protein-functionalized SMBs for ligand fishing,prompting its usage on drug screening and target finding.
基金supported by the National Natural Science Foundation of China(Nos.81930122 and U20A20361)the National Key Research and Development Project of China(No.2021YFC2102101).
文摘Conjugate vaccines represent one of the most effective means for controlling the occurrence of bacterial diseases.Although nanotechnology has been greatly applied in the field of vaccines,it is seldom used for conjugate vaccine research because it is very difficult to connect polysaccharides and nanocarriers.In this work,an orthogonal and modular biosynthesis method was used to produce nanoconjugate vaccines using the SpyTag/SpyCatcher system.When SpyTag/SpyCatcher system is combined with protein glycosylation technology,bacterial O-polysaccharide obtained from Shigela flexneri 2a can be conjugated onto the surfaces of different virus-like particles(VLPs)in a biocompatible and controlled manner.After confirming the excellent lymph node targeting and humoral immune activation abilities,these nanoconjugate vaccines further induced efficient prophylactic effects against infection in a mouse model.These results demonstrated that natural polysaccharide antigens can be easily connected to VLPs to prepare highly efficient nanoconjugate vaccines.To the best of the researchers1 knowledge,this is the first time VLP-based nanoconjugate vaccines are produced efficiently,and this strategy could be applied to develop various pathogenic nanoconjugate vaccines.
基金the Ministry of Science and Technology to F.S.(No.2020YFA0908100)from the National Natural Science Foundation of China Excellent Young Scientists Fund to F.S.(No.22122707)+8 种基金from National Natural Science Foundation of China Youth fund to S.Z.K.(No.21907003)from the National Natural Science Foundation of China from Guangdong Natural Science Foundation to F.S.(No.2019A1515011691)S.Z.K.(No.2020A1515011429)from the Science,Technology,and Innovation Commission of Shenzhen Municipality to F.S.(Shenzhen-Hong Kong-Macao S&T Program(Category C)#SGDX2020110309460101Key Research Program#JCYJ20200109141241950Basic Research Program#JCYJ20190813094601656)from the Research Grants Council of Hong Kong SAR Government to F.S.(General Research Fund#16103519 and#16103421Theme-based Research Scheme#T13-602/21-N)from the State Key Laboratory of Molecular Neuroscience,HKUST,China,and in part from the Innovation and Technology Commission(ITCPD/17-9)are acknowledged.
文摘The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has wreaked havoc around the globe,with no end in sight.The rapid emergence of viral mutants,marked by rapid transmission and effective immune evasion,has also posed unprecedented challenges for vaccine development,not least in its speed,mass production,and distribution.Here we report a versatile“plug-and-display”strategy for creating protein vaccines,including those against malaria parasites and SARS-CoV-2,through the combined use of the intrinsically disordered protein ligase SpyStapler and computationally designed viral-like particles.The resulting protein nanoparticles harboring multiple antigens induce potent neutralizing antibody responses in mice,substantially stronger than those induced by the corresponding free antigens.This modular vaccine design enabled by SpyStapler furnishes us with a new weapon for combatting infectious diseases.
