Progress 05/15/19 to 08/20/21
Outputs
Target Audience:Two PhD students who are supported by this project got regular training and education on related knowledge and lab techniques. The PIs also instructed the graduate students in their classes by presenting this project as an example of hybrid nanomaterials for nanobiotechnology and environmental remodation. The PI also presented the research to faculty members and postdoc researchers at the University of Notre Dame Energy Center and Environmental Change Initiative. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project provided a training opportunity for two PhD students in two departments: environmental engineering and chemistry. The project also provides training opportunities for a research associate who had a master degree. The research technician gained research experience through this project and has been admitted to the Department of Chemistry and Biochemistry to pursue PhD study in Fall 2020. The project involved one undergraduate student in Environmental Engineering and provided training opportunities for the student to gain research experience and learn about P recovery. How have the results been disseminated to communities of interest?One manuscript has been prepared and submitted to the journal "ACS Applied Materials and Interfaces". In this paper, we for the first time developed a novel magnetic nanoparticle (MNP) platform for covalent protein immobilization via SpyTag/SpyCatcher chemistry. The synthesis of SpyTag-functionalized MNPs is facile for easy scaleup; and the conjugation of SpyCatcher-fused protein onto the MNP is efficient and specific, not influenced by the presence of Tween 20 detergent and BSA. Both fluorescence microscopy and spectroscopy characterizations confirmed that the surface-immobilized EGFP retained strong fluorescence intensity and demonstrated significantly improved fluorescent stability compared to free proteins. The EGFP-conjugated MNPs exhibited high colloidal stability and can be efficiently separated from solution under magnetic field to achieve a convenient recovery and reuse of proteins. We expect this developed MNP platform will provide a universal and modular water-dispersible protein immobilization approach that allows easy separation and reusability of proteins, including phosphate binding proteins. The PI presented the project idea and initial results in the luncheon seminar hosted by Notre Dame Energy (ND Energy), which is an interdisciplinary center that fosters research relevant to energy and sustainability. The PI also presented the project in the Environmental Change Initiative (ECI) at ND, which reached around fifty faculty members who work on issues including global warming, climate change, and alterations to land, water and air that are threatening human and environmental welfare. The PIs and the graduate students on this project plans to disseminate the research results to broader audience in the communities of interest through conferences when pandemic situation gets better. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In this project funded by the Agricultural and Food Systems program, United States Department of Agriculture, Professors Wei and Gao from the University of Notre Dame are developing a new class of biomimetic material for selective capture and efficient recovery of phosphate from agricultural and animal waste streams. Phosphorus (P) is one of the essential nutrient elements for the growth of plants and animals, being critical to sustain the global food supply, but this element is in short supply in nature. Currently, about 80% of the mined phosphorus is applied to agricultural fields, but only about 40% finds its way into harvested crops and 16% is consumed as human food. Most P is lost in the discharge of raw or treated municipal, agricultural and industrial waste streams, which should and could be recovered. One of the biggest challenges for P recovery lies in selective separation of phosphate over various other ions present in aqueous systems. This project will create a new class of hybrid nanomaterials that contain functionalized biomolecules for selective capture of phosphate and magnetic nanoparticles in the core for subsequent recovery of the phosphate. The study is the first to engineer a recombinant phosphate binding protein (PBP) linked onto a magnetic nanoparticle platform via an efficient covalent Spy chemistry, i.e., a covalent isopeptide bond between an aspartic acid residue of SpyTag peptide (13 amino acids) and a lysine residue of SpyCatcher domain (~ 13 KDa). Establishment of such a recombinant phosphate binding protein expression system is highly valuable for scalable and cost-effective protein production and downstream immobilization. By immobilizing the PBP on a magnetic nanoparticle to construct hybrid nanomaterials, the study will identify effective strategies to enhance phosphate recovery efficiency and stability of the material in reuse. A new technology that jointly meets the needs of efficient phosphorus removal and recovery as well as reuse of the material can be developed. In this project period, our accomplished research activities are summarized below: Specific objective 1. developing a modular MNP platform that can achieve easy separation under magnetic force and efficient conjugation of proteins onto the surface. 1) We developed an efficient and scalable synthesis method to construct the core-shell structured MNP platform that contained three domains, a Fe3O4 nanocluster core, a SiO2 middle shell, and a surface-grafted polymer corona. 2) Quaternization of the amine groups on polymer arms not only introduced permanent positive charge onto the polymer shell, but also introduced numerous reactive allyl groups for subsequent thiol-ene reaction with cysteine-terminated SpyTag peptide. 3) A model SpyCatcher-fused fluorescent protein, EGFP-SpyCatcher, was successfully conjugated onto the SpyTag-functionalized MNP platform without adding enzymes or chemical cross-linkers. No activity loss was observed on EGFP after conjugation due to the site-selective nature of Spy chemistry. The conjugation of EGFP on MNP was highly specific and robust, which was not affected by the presence of other proteins and detergents, such as Bovine serum albumin (BSA) and Tween 20 detergent. 4) The MNP platform was demonstrated to be protective to the conjugated EGFP and significantly improved the shelf life of immobilized proteins. With the above research results, we have successfully accomplished our objective 1 by developing a modular SpyTag-functionalized MNP platform, that enables the conjugation of various SpyCatcher-fused functional proteins, including PBPs. One manuscript has been submitted to ACS Applied Materials and Interfaces. The manuscript draft is attached. Specific objective 2. characterize the MNP platform as a generic and modular water-dispersible protein immobilization approach that allows easy separation and reusability of proteins. 1) Three different SpyCatcher-fused fluorescent proteins, including EGFP-SpyCatcher, RFP-SpyCatcher, and YFP-SpyCatcher, were successfully conjugated onto the SpyTag-functionalized MNP. The maximal protein loading on MNP and SpyTag utilization efficiency were achieved at relatively low SpyTag:SpCatcher molar ratio (i.e., 2ST:1SC). 2) SpyTag-functionalized MNP showed high specificity to conjugated EGFP-SpyCatcher or RFP-SpyCatcher from complex cell lysate. 3) Modular assembly of multiple proteins on MNP was achieved either from the mixture of two different proteins or their cell lysate mixture. Each protein loading on MNP was tunable by varying their molar quantities. 4) The MNP platform significantly improved the long-term stability of immobilized proteins and their robustness to extreme conditions, such as high temperature and freeze-thaw process. With the above research results, we have demonstrated that the MNP platform can enable specific separation of proteins from complex cell lysate and allow modular immobilization of multiple proteins. One manuscript is is in preparation. The summary of results under this objective is attached. Specific objective 3. conjugating PBP onto MNP platform and optimizing the MNP-PBP through comprehensive understanding of structure-function relationships, We created expression plasmids for producing the recombinant fusion phosphate binding protein, specifically SpyCatcher-fused PstS from E. coli (i.e. PstS) and successfully express the fusion protein in the E. coli host cell. We established the protocol to isolate and purify recombinant phosphate binding protein SpyCatcher-PstS with a yield of 200 mg protein at bench scale. We characterized the function and stability SpyCatcher-fused PstS protein in adsorption and desorption of phosphate in buffer systems. Results demonstrated that the fusion protein can adsorb and desorb phosphate by controlling pH of the solution. The protein is stable at room temperature (with relative activity remaining above 80% after a month experimental period). With the above research accomplishments, we have created the magnetic nanoparticles with SpyTag. Next goal is to conjugate the SpyCatcher-PstS with the engineered magnetic nanoparticle. Introduce SpyCatcher-fused phosphate binding protein SpyCatcher-PstS onto SpyTag-functionalized MNP platform to obtain PstS-conjugated MNP. Characterize the function and stability of the PstS-conjugated MNP regarding their adsorption and desorption of phosphate under various pH solutions, the collidable stability and magnetic separation of MNP under various pH dispersions. Specific objective 4. determining the performance of MN-PBP in terms of P recovery efficiency, stability in reuse and robustness under application relevant conditions and identifying strategy for optimization. We aim to work on this objective in the remaining project period.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2022
Citation:
Xiuyu Jin, Quanhui Ye, Chien-Wei Wang, Ying Wu, Kangling Ma, Sihan Yu, Na Wei and Haifeng Gao, Magnetic
Nanoplatform for Covalent Protein Immobilization Based on Spy Chemistry, submitted to ACS Applied Materials &
Interfaces
|
Progress 05/15/20 to 05/14/21
Outputs
Target Audience:Two PhD students who are supported by this project got regular training and education on related knowledge and lab techniques. The PIs also instructed the graduate students in their classes by presenting this project as an example of hybrid nanomaterials for nanobiotechnology and environmental remodation. The PI also presented the research to faculty members and postdoc researchers at the University of Notre Dame Energy Center and Environmental Change Initiative. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project provided a training opportunity for two PhD students in two departments: environmental engineering and chemistry. The project also provides training opportunities for a research associate who had a master degree. The research technician gained research experience through this project and has been admitted to the Department of Chemistry and Biochemistry to pursue PhD study in Fall 2020. The project involved one undergraduate student in Environmental Engineering and provided training opportunities for the student to gain research experience and learn about P recovery. How have the results been disseminated to communities of interest?One manuscript has been prepared and submitted to the journal "ACS Applied Materials and Interfaces". In this paper, we for the first time developed a novel magnetic nanoparticle (MNP) platform for covalent protein immobilization via SpyTag/SpyCatcher chemistry. The synthesis of SpyTag-functionalized MNPs is facile for easy scaleup; and the conjugation of SpyCatcher-fused protein onto the MNP is efficient and specific, not influenced by the presence of Tween 20 detergent and BSA. Both fluorescence microscopy and spectroscopy characterizations confirmed that the surface-immobilized EGFP retained strong fluorescence intensity and demonstrated significantly improved fluorescent stability compared to free proteins. The EGFP-conjugated MNPs exhibited high colloidal stability and can be efficiently separated from solution under magnetic field to achieve a convenient recovery and reuse of proteins. We expect this developed MNP platform will provide a universal and modular water-dispersible protein immobilization approach that allows easy separation and reusability of proteins, including phosphate binding proteins. The PI presented the project idea and initial results in the luncheon seminar hosted by Notre Dame Energy (ND Energy), which is an interdisciplinary center that fosters research relevant to energy and sustainability. The PI also presented the project in the Environmental Change Initiative (ECI) at ND, which reached around fifty faculty members who work on issues including global warming, climate change, and alterations to land, water and air that are threatening human and environmental welfare. The PIs and the graduate students on this project plans to disseminate the research results to broader audience in the communities of interest through conferences when pandemic situation gets better. What do you plan to do during the next reporting period to accomplish the goals? Conjugate the SpyCatcher-PstS protein onto the engineered magnetic nanoparticle with SpyTag, to create the proposed material MN-PBP Characterize the structure-function relationships of the MN-PBP and optimize the material at the molecular level to enhance its performance in terms of P recovery efficiency and stability. Assess the robustness of the MN-PBP under the environmentally relevant and application relevant conditions, which will provide a basis for further optimization.
Impacts
What was accomplished under these goals?
In this project funded by the Agricultural and Food Systems program, United States Department of Agriculture, Professors Wei and Gao from the University of Notre Dame are developing a new class of biomimetic material for selective capture and efficient recovery of phosphate from agricultural and animal waste streams. Phosphorus (P) is one of the essential nutrient elements for the growth of plants and animals, being critical to sustain the global food supply, but this element is in short supply in nature. Currently, about 80% of the mined phosphorus is applied to agricultural fields, but only about 40% finds its way into harvested crops and 16% is consumed as human food. Most P is lost in the discharge of raw or treated municipal, agricultural and industrial waste streams, which should and could be recovered. One of the biggest challenges for P recovery lies in selective separation of phosphate over various other ions present in aqueous systems. This project will create a new class of hybrid nanomaterials that contain functionalized biomolecules for selective capture of phosphate and magnetic nanoparticles in the core for subsequent recovery of the phosphate. The study is the first to engineer a recombinant phosphate binding protein (PBP) linked onto a magnetic nanoparticle platform via an efficient covalent Spy chemistry, i.e., a covalent isopeptide bond between an aspartic acid residue of SpyTag peptide (13 amino acids) and a lysine residue of SpyCatcher domain (~ 13 KDa). Establishment of such a recombinant phosphate binding protein expression system is highly valuable for scalable and cost-effective protein production and downstream immobilization. By immobilizing the PBP on a magnetic nanoparticle to construct hybrid nanomaterials, the study will identify effective strategies to enhance phosphate recovery efficiency and stability of the material in reuse. A new technology that jointly meets the needs of efficient phosphorus removal and recovery as well as reuse of the material can be developed. In this project period, our accomplished research activities are summarized below: Specific objective 1. developing a modular MNP platform that can achieve easy separation under magnetic force and efficient conjugation of proteins onto the surface. 1) We developed an efficient and scalable synthesis method to construct the core-shell structured MNP platform that contained three domains, a Fe3O4 nanocluster core, a SiO2 middle shell, and a surface-grafted polymer corona. 2) Quaternization of the amine groups on polymer arms not only introduced permanent positive charge onto the polymer shell, but also introduced numerous reactive allyl groups for subsequent thiol-ene reaction with cysteine-terminated SpyTag peptide. 3) A model SpyCatcher-fused fluorescent protein, EGFP-SpyCatcher, was successfully conjugated onto the SpyTag-functionalized MNP platform without adding enzymes or chemical cross-linkers. No activity loss was observed on EGFP after conjugation due to the site-selective nature of Spy chemistry. The conjugation of EGFP on MNP was highly specific and robust, which was not affected by the presence of other proteins and detergents, such as Bovine serum albumin (BSA) and Tween 20 detergent. 4) The MNP platform was demonstrated to be protective to the conjugated EGFP and significantly improved the shelf life of immobilized proteins. With the above research results, we have successfully accomplished our objective 1 by developing a modular SpyTag-functionalized MNP platform, that enables the conjugation of various SpyCatcher-fused functional proteins, including PBPs. One manuscript has been submitted to ACS Applied Materials and Interfaces. The manuscript draft is attached. Specific objective 2. characterize the MNP platform as a generic and modular water-dispersible protein immobilization approach that allows easy separation and reusability of proteins. 1) Three different SpyCatcher-fused fluorescent proteins, including EGFP-SpyCatcher, RFP-SpyCatcher, and YFP-SpyCatcher, were successfully conjugated onto the SpyTag-functionalized MNP. The maximal protein loading on MNP and SpyTag utilization efficiency were achieved at relatively low SpyTag:SpCatcher molar ratio (i.e., 2ST:1SC). 2) SpyTag-functionalized MNP showed high specificity to conjugated EGFP-SpyCatcher or RFP-SpyCatcher from complex cell lysate. 3) Modular assembly of multiple proteins on MNP was achieved either from the mixture of two different proteins or their cell lysate mixture. Each protein loading on MNP was tunable by varying their molar quantities. 4) The MNP platform significantly improved the long-term stability of immobilized proteins and their robustness to extreme conditions, such as high temperature and freeze-thaw process. With the above research results, we have demonstrated that the MNP platform can enable specific separation of proteins from complex cell lysate and allow modular immobilization of multiple proteins. One manuscript is is in preparation. The summary of results under this objective is attached. Specific objective 3. conjugating PBP onto MNP platform and optimizing the MNP-PBP through comprehensive understanding of structure-function relationships, We created expression plasmids for producing the recombinant fusion phosphate binding protein, specifically SpyCatcher-fused PstS from E. coli (i.e. PstS) and successfully express the fusion protein in the E. coli host cell. We established the protocol to isolate and purify recombinant phosphate binding protein SpyCatcher-PstS with a yield of 200 mg protein at bench scale. We characterized the function and stability SpyCatcher-fused PstS protein in adsorption and desorption of phosphate in buffer systems. Results demonstrated that the fusion protein can adsorb and desorb phosphate by controlling pH of the solution. The protein is stable at room temperature (with relative activity remaining above 80% after a month experimental period). With the above research accomplishments, we have created the magnetic nanoparticles with SpyTag. Next goal is to conjugate the SpyCatcher-PstS with the engineered magnetic nanoparticle. Introduce SpyCatcher-fused phosphate binding protein SpyCatcher-PstS onto SpyTag-functionalized MNP platform to obtain PstS-conjugated MNP. Characterize the function and stability of the PstS-conjugated MNP regarding their adsorption and desorption of phosphate under various pH solutions, the collidable stability and magnetic separation of MNP under various pH dispersions. Specific objective 4. determining the performance of MN-PBP in terms of P recovery efficiency, stability in reuse and robustness under application relevant conditions and identifying strategy for optimization. We aim to work on this objective in the remaining project period.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2021
Citation:
Xiuyu Jin, Quanhui Ye, Chien-Wei Wang, Ying Wu, Kangling Ma, Sihan Yu, Na Wei and Haifeng Gao, Magnetic Nanoplatform for Covalent Protein Immobilization Based on Spy Chemistry, submitted to ACS Applied Materials & Interfaces
|
Progress 05/15/19 to 05/14/20
Outputs
Target Audience:The project PIseducated two PhD students and one research technician on the project related knowledge and trained them on the related lab techniques. The PIs also instructed the graduate students in their classes by presenting this project as an example of nanobiotechnology. The PI also presented the research to faculty members and postdoc researchers at the University of Notre Dame Energy Center and Environmental Change Initiative. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project provided a training opportunity for two PhD students and a research technician in two departments: environmental engineering and chemistry. The project also provides training opportunities for a research associate who had a master degree. The research technician gained research experience through this project and has been admitted to the Department of Chemistry and Biochemistry to pursue PhD study in Fall 2020. The project involved one undergraduate student in Environmental Engineering and provided training opportunity for the student to gain research experience and learn about P recovery. How have the results been disseminated to communities of interest?The PI presented the project idea and initial results in the luncheon seminar hosted by Notre Dame Energy (ND Energy), which is an interdisciplinary center that fosters research relevant to energy and sustainability. The PI also presented the project in the Environmental Change Initiative (ECI) at ND, which reached around fifty faculty members who work on issues including global warming, climate change, and alterations to land, water and air that are threatening human and environmental welfare. The coPI, together with Prof. Matthew Webber (ND), Prof. Brett Savoie(Purdue) and Prof. Jianguo Mei (Purdue) successfully organized the 6th Annual NotreDame-Purdue Symposium on Soft Matter & Polymers and Poster Session on Saturday,September 21, 2019 at Purdue University. This symposium in 2019 covered the topics of synthesis, characterization and computational calculation in the broadly defined synthetic soft materials, with focus on their applications in energy and biomaterials. The symposium included faculty talks,student poster session, along with networking social time. Eight students by the end of symposium received poster award. What do you plan to do during the next reporting period to accomplish the goals? Conjugate the SpyCatcher-PstS protein onto the engineered magnetic nanoparticle with SpyTag, to create the proposed material MN-PBP Characterize the structure-function relationships of the MN-PBP and optimize the material at the molecular level to enhance its performance in terms of P recovery efficiency and stability. Assess the robustness of the MN-PBP under the environmentally relevant and application relevant conditions, which will provide a basis for further optimization.
Impacts
What was accomplished under these goals?
In this project funded by the Agricultural and Food Systems program, United States Department of Agriculture, Professors Wei and Gao from the University of Notre Dame are developing a new class of biomimetic material for selective capture and efficient recovery of phosphate from agricultural and animal waste streams. Phosphorus (P) is one of the essential nutrient elements for the growth of plants and animals, being critical to sustain the global food supply, but this element is in short supply in nature. Currently, about 80% of the mined phosphorus is applied to agricultural fields, but only about 40% finds its way into harvested crops and 16% is consumed as human food. Most P is lost in the discharge of raw or treated municipal, agricultural and industrial waste streams, which should and could be recovered. One of the biggest challenges for P recovery lies in selective separation of phosphate over various other ions present in aqueous systems. This project will create a new class of hybrid nanomaterials that contain functionalized biomolecules for selective capture of phosphate and magnetic nanoparticles in the core for subsequent recovery of the phosphate. The study is the first to engineer a recombinant phosphate binding protein (PBP) linked with a bioaffinity tag molecule (e.g. SpyCatcher-SpyTag system). Establishment of such a recombinant phosphate binding protein expression system is highly valuable for scalable and cost-effective protein production and downstream immobilization. By immobilizing the PBP on magnetic nanoparticles to construct MN-PBP hybrids, the study will identify effective strategies to enhance phosphate recovery efficiency and stability of the material in reuse. A new technology that jointly meets the needs of efficient phosphorus removal and recovery as well as reuse of the material can be developed. In this project period, our accomplished research activities are summarized below: Specific objective 1. Generate functional recombinant PBP and quantify its phosphate binding properties Create expression plasmids for producing the recombinant fusion phosphate binding protein, specifically SpyCatcher fused PBP from E. coli (i.e. PstS) and successfully express the fusion protein in the E. coli host cell. Establish the protocol to isolate and purify recombinant phosphate binding protein SpyCatcher-PstS with a yield of 200 mg protein at bench scale. Characterize functionality and stability of the fusion protein in adsorption and desorption of phosphate in buffer systems. Results demonstrate that the fusion protein can adsorb and desorb phosphate by controlling pH of the solution. The protein is stable at room temperature (with relative activity remained above 80% after a month experimental period) With the above research accomplishments, we have successfully achieved the objective 1. Specific objective 2. Construct and optimize the MN-PBP through comprehensive understanding of structure-function relationships Develop the synthesis procedures for magnetic nanoparticles that contain supermagnetic core, inert silica shell and polyelectrolyte dangling hair to be dispersible in water. Introduce chelating ligand peptide SpyTag onto the polyelectrolyte polymer hair for next-step complexation with the complementary receptor SpyCatcher-fused PBP. Characterize the composition and structure of the magnetic nanoparticles with or without the surface conjugated SpyTag. With the above research accomplishments, we have created the magnetic nanoparticles with SpyTag. Next goal is to conjugate the SpyCatcher-PstS with the engineered magnetic nanoparticle. Specific objective 3. Determine the performance of MN-PBP in terms of P recovery efficiency, stability in reuse and robustness under application relevant conditions and identify strategy for optimization. We aim to work on this objective in the remaining project period.
Publications
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