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
Unlike small molecules,the topological complexity of macromolecules remains largely unexplored due to the huge synthetic challenge.Herein,we report the development of orthogonal active templates for concise and select...Unlike small molecules,the topological complexity of macromolecules remains largely unexplored due to the huge synthetic challenge.Herein,we report the development of orthogonal active templates for concise and selective synthesis of protein[n]heterocatenanes toward protein olympiadanes.An active template(AT-Snoop)was first developed based on the isopeptide-bond-forming RrgA domain with comparable efficiency and excellent orthogonality to the previously reported active template(AT-Spy)based on the CnaB2 domain.Their combination facilitated the selective synthesis of protein[n]catenanes from multiple components in one step and the resulting structures were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis,size exclusion chromatography,liquid chromatography-mass spectrometry,and proteolytic digestion experiments.The results offered a promising solution to tackling the daunting challenge of precision synthesis of protein olympiadane with five distinct ring components.Not only did the success provide new tools forproteintopologyengineeringbutalsospurred and fueled the future exploitation of topology-related functional benefits in protein science.展开更多
The expansion of protein topological diversity requires new and efficient synthetic tools.Herein,we report the second and third generations of the SpyStapler-mediated SpyTag/BDTag ligation system for the efficient syn...The expansion of protein topological diversity requires new and efficient synthetic tools.Herein,we report the second and third generations of the SpyStapler-mediated SpyTag/BDTag ligation system for the efficient synthesis of 4-arm star proteins and the repurposing of the third generation as an active template to enable the synthesis of higher-order protein[n]catenanes(n=3,4,and 5).SpyStapler003 has a higher affinity to its cognate SpyTag and BDTag reactive pairs relative to the original SpyStapler.Hence,it can overcome much more profound steric hindrance in protein ligation and improve the efficiency of the resulting active template tool to facilitate the construction of radial protein[n]catenanes.Various proteins of interest,such as dihydrofolate reductase and the nanobody KN035,can be modularly incorporated into the[n]catenanes with intact activity.Combination of passive and active template strategies gives rise to linear protein[4]catenanes,which further expands the current topological diversity.Moreover,higher-order protein catenation not only leads to enhanced thermal stability and proteolytic resistance but also higher affinity of the nanobody via multivalent effects.Our study provides tools useful for bioconjugation and new topological protein scaffolds for the multivalent display of enzymes and antibodies.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
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
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the National Natural Science Foundation of China(grant nos.21991132,21925102,92056118,22101010,22201017,and 22201016)the National Key R&D Program of China(grant no.2020YFA0908100)+1 种基金Beijing National Laboratory for Molecular Sciences(grant no.BNLMS-CXXM-202006)supported by the National Center for Protein Sciences at Peking University.
文摘Unlike small molecules,the topological complexity of macromolecules remains largely unexplored due to the huge synthetic challenge.Herein,we report the development of orthogonal active templates for concise and selective synthesis of protein[n]heterocatenanes toward protein olympiadanes.An active template(AT-Snoop)was first developed based on the isopeptide-bond-forming RrgA domain with comparable efficiency and excellent orthogonality to the previously reported active template(AT-Spy)based on the CnaB2 domain.Their combination facilitated the selective synthesis of protein[n]catenanes from multiple components in one step and the resulting structures were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis,size exclusion chromatography,liquid chromatography-mass spectrometry,and proteolytic digestion experiments.The results offered a promising solution to tackling the daunting challenge of precision synthesis of protein olympiadane with five distinct ring components.Not only did the success provide new tools forproteintopologyengineeringbutalsospurred and fueled the future exploitation of topology-related functional benefits in protein science.
基金support from the National Natural Science Foundation of China(grant nos.21991132,21925102,92056118,22101010,22201016,and 22201017)the National Key R&D Program of China(grant no.2020YFA0908100)the Beijing National Laboratory for Molecular Sciences(grant no.BNLMSCXXM-202006)。
文摘The expansion of protein topological diversity requires new and efficient synthetic tools.Herein,we report the second and third generations of the SpyStapler-mediated SpyTag/BDTag ligation system for the efficient synthesis of 4-arm star proteins and the repurposing of the third generation as an active template to enable the synthesis of higher-order protein[n]catenanes(n=3,4,and 5).SpyStapler003 has a higher affinity to its cognate SpyTag and BDTag reactive pairs relative to the original SpyStapler.Hence,it can overcome much more profound steric hindrance in protein ligation and improve the efficiency of the resulting active template tool to facilitate the construction of radial protein[n]catenanes.Various proteins of interest,such as dihydrofolate reductase and the nanobody KN035,can be modularly incorporated into the[n]catenanes with intact activity.Combination of passive and active template strategies gives rise to linear protein[4]catenanes,which further expands the current topological diversity.Moreover,higher-order protein catenation not only leads to enhanced thermal stability and proteolytic resistance but also higher affinity of the nanobody via multivalent effects.Our study provides tools useful for bioconjugation and new topological protein scaffolds for the multivalent display of enzymes and antibodies.
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.