Protein Modification Employing Non-Canonical Amino Acids to Prepare SUMOylation Detecting Bioconjugates
Protein modification with non-canonical amino acids (ncAAs) represents a useful technology to afford homogenous samples of bioconjugates with site-specific modification. This technique can be directly applied to the detection of aberrant SUMOylation patterns, which are often indicative of disease st...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-12-01
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| Series: | Pharmaceutics |
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| Online Access: | https://www.mdpi.com/1999-4923/14/12/2826 |
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| author | Alexander C. Williard Hannah J. Switzer Christina A. Howard Rui Yin Brent L. Russell Ritwik Sanyal Shaun Yu Trinity M. Myers Brian M. Flood Oliver Kerscher Douglas D. Young |
| author_facet | Alexander C. Williard Hannah J. Switzer Christina A. Howard Rui Yin Brent L. Russell Ritwik Sanyal Shaun Yu Trinity M. Myers Brian M. Flood Oliver Kerscher Douglas D. Young |
| author_sort | Alexander C. Williard |
| collection | DOAJ |
| description | Protein modification with non-canonical amino acids (ncAAs) represents a useful technology to afford homogenous samples of bioconjugates with site-specific modification. This technique can be directly applied to the detection of aberrant SUMOylation patterns, which are often indicative of disease states. Modified SUMO-trapping proteins, consisting of a catalytically inactive ULP1 fragment (UTAG) fused to the maltose-binding protein MBP, are useful reagents for the binding and labeling of SUMOylated proteins. Mutation of this UTAG fusion protein to facilitate amber suppression technologies for the genetic incorporation of ncAAs was assessed to provide a functional handle for modification. Ultimately, two sites in the maltose-binding protein (MBP) fusion were identified as ideal for incorporation and bioconjugation without perturbation to the SUMO-trapping ability of the UTAG protein. This functionality was then employed to label SUMOylated proteins in HeLa cells and demonstrate their enrichment in the nucleus. This modified UTAG-MBP-ncAA protein has far-reaching applications for both diagnostics and therapeutics. |
| first_indexed | 2024-03-09T15:57:57Z |
| format | Article |
| id | doaj.art-fdeb925ff51f4b61b7dff9b3e8228671 |
| institution | Directory Open Access Journal |
| issn | 1999-4923 |
| language | English |
| last_indexed | 2024-03-09T15:57:57Z |
| publishDate | 2022-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Pharmaceutics |
| spelling | doaj.art-fdeb925ff51f4b61b7dff9b3e82286712023-11-24T17:22:41ZengMDPI AGPharmaceutics1999-49232022-12-011412282610.3390/pharmaceutics14122826Protein Modification Employing Non-Canonical Amino Acids to Prepare SUMOylation Detecting BioconjugatesAlexander C. Williard0Hannah J. Switzer1Christina A. Howard2Rui Yin3Brent L. Russell4Ritwik Sanyal5Shaun Yu6Trinity M. Myers7Brian M. Flood8Oliver Kerscher9Douglas D. Young10Department of Chemistry, William & Mary, Williamsburg, VA 23185, USADepartment of Chemistry, William & Mary, Williamsburg, VA 23185, USADepartment of Chemistry, William & Mary, Williamsburg, VA 23185, USADepartment of Chemistry, William & Mary, Williamsburg, VA 23185, USADepartment of Chemistry, William & Mary, Williamsburg, VA 23185, USADepartment of Chemistry, William & Mary, Williamsburg, VA 23185, USADepartment of Chemistry, William & Mary, Williamsburg, VA 23185, USADepartment of Chemistry, William & Mary, Williamsburg, VA 23185, USADepartment of Chemistry, William & Mary, Williamsburg, VA 23185, USADepartment of Biology, William & Mary, Williamsburg, VA 23185, USADepartment of Chemistry, William & Mary, Williamsburg, VA 23185, USAProtein modification with non-canonical amino acids (ncAAs) represents a useful technology to afford homogenous samples of bioconjugates with site-specific modification. This technique can be directly applied to the detection of aberrant SUMOylation patterns, which are often indicative of disease states. Modified SUMO-trapping proteins, consisting of a catalytically inactive ULP1 fragment (UTAG) fused to the maltose-binding protein MBP, are useful reagents for the binding and labeling of SUMOylated proteins. Mutation of this UTAG fusion protein to facilitate amber suppression technologies for the genetic incorporation of ncAAs was assessed to provide a functional handle for modification. Ultimately, two sites in the maltose-binding protein (MBP) fusion were identified as ideal for incorporation and bioconjugation without perturbation to the SUMO-trapping ability of the UTAG protein. This functionality was then employed to label SUMOylated proteins in HeLa cells and demonstrate their enrichment in the nucleus. This modified UTAG-MBP-ncAA protein has far-reaching applications for both diagnostics and therapeutics.https://www.mdpi.com/1999-4923/14/12/2826bioconjugatesnon-canonical amino acidsSUMOprotein modification |
| spellingShingle | Alexander C. Williard Hannah J. Switzer Christina A. Howard Rui Yin Brent L. Russell Ritwik Sanyal Shaun Yu Trinity M. Myers Brian M. Flood Oliver Kerscher Douglas D. Young Protein Modification Employing Non-Canonical Amino Acids to Prepare SUMOylation Detecting Bioconjugates Pharmaceutics bioconjugates non-canonical amino acids SUMO protein modification |
| title | Protein Modification Employing Non-Canonical Amino Acids to Prepare SUMOylation Detecting Bioconjugates |
| title_full | Protein Modification Employing Non-Canonical Amino Acids to Prepare SUMOylation Detecting Bioconjugates |
| title_fullStr | Protein Modification Employing Non-Canonical Amino Acids to Prepare SUMOylation Detecting Bioconjugates |
| title_full_unstemmed | Protein Modification Employing Non-Canonical Amino Acids to Prepare SUMOylation Detecting Bioconjugates |
| title_short | Protein Modification Employing Non-Canonical Amino Acids to Prepare SUMOylation Detecting Bioconjugates |
| title_sort | protein modification employing non canonical amino acids to prepare sumoylation detecting bioconjugates |
| topic | bioconjugates non-canonical amino acids SUMO protein modification |
| url | https://www.mdpi.com/1999-4923/14/12/2826 |
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