Protein engineering for covalent immobilization and enhanced stability through incorporation of multiple noncanonical amino acids
In this study, we demonstrate the application of multiple functional properties of proteins generated through coupling of residue-specific and site-specific incorporation method. With green fluorescent protein (GFP) as a model protein, we constructed multifunctional GFP through sitespecific incorpor...
| Main Authors: | , , , |
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| Other Authors: | |
| Format: | Journal Article |
| Language: | English |
| Published: |
2017
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| Online Access: | https://hdl.handle.net/10356/86561 http://hdl.handle.net/10220/44074 |
| _version_ | 1826120053439856640 |
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| author | Deepankumar, Kanagavel Prabhu, Nadarajan Saravanan Kim, June-Hyung Yun, Hyungdon |
| author2 | School of Materials Science & Engineering |
| author_facet | School of Materials Science & Engineering Deepankumar, Kanagavel Prabhu, Nadarajan Saravanan Kim, June-Hyung Yun, Hyungdon |
| author_sort | Deepankumar, Kanagavel |
| collection | NTU |
| description | In this study, we demonstrate the application of multiple functional properties of proteins generated through coupling of residue-specific and site-specific incorporation method. With green fluorescent protein (GFP) as a model protein, we constructed multifunctional GFP through sitespecific incorporation of L-3,4-dihydroxyphenylalanine (DOPA) and residue-specific incorporation of (2S, 4S)-4- fluoroproline (4S-FP) or L-homopropargylglycine (hpg). Fluorescence analysis revealed a conjugation efficiency of approximately 20% for conjugation of DOPA-containing variants GFPdopa, GFPdp[4S-FP], and GFPdphpg onto chitosan. While incorporation of 4S-FP improved protein folding and stability, hpg incorporation into GFP allowed conjugation with fluorescent dye/polyethylene glycol (PEG). In addition, the modification of GFPhpg and GFPdphpg with PEG through Cu(I)-catalyzed click reaction increased protein thermal stability by about two-fold of the wild-type GFP. |
| first_indexed | 2024-10-01T05:10:15Z |
| format | Journal Article |
| id | ntu-10356/86561 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T05:10:15Z |
| publishDate | 2017 |
| record_format | dspace |
| spelling | ntu-10356/865612020-06-01T10:21:21Z Protein engineering for covalent immobilization and enhanced stability through incorporation of multiple noncanonical amino acids Deepankumar, Kanagavel Prabhu, Nadarajan Saravanan Kim, June-Hyung Yun, Hyungdon School of Materials Science & Engineering Multiple noncanonical amino acids Fluoroproline In this study, we demonstrate the application of multiple functional properties of proteins generated through coupling of residue-specific and site-specific incorporation method. With green fluorescent protein (GFP) as a model protein, we constructed multifunctional GFP through sitespecific incorporation of L-3,4-dihydroxyphenylalanine (DOPA) and residue-specific incorporation of (2S, 4S)-4- fluoroproline (4S-FP) or L-homopropargylglycine (hpg). Fluorescence analysis revealed a conjugation efficiency of approximately 20% for conjugation of DOPA-containing variants GFPdopa, GFPdp[4S-FP], and GFPdphpg onto chitosan. While incorporation of 4S-FP improved protein folding and stability, hpg incorporation into GFP allowed conjugation with fluorescent dye/polyethylene glycol (PEG). In addition, the modification of GFPhpg and GFPdphpg with PEG through Cu(I)-catalyzed click reaction increased protein thermal stability by about two-fold of the wild-type GFP. 2017-11-24T06:27:22Z 2019-12-06T16:24:45Z 2017-11-24T06:27:22Z 2019-12-06T16:24:45Z 2017 2017 Journal Article Deepankumar, K., Prabhu, N. S., Kim, J.-H., & Yun, H. (2017). Protein engineering for covalent immobilization and enhanced stability through incorporation of multiple noncanonical amino acids. Biotechnology and Bioprocess Engineering, 22(3), 248-255. 1226-8372 https://hdl.handle.net/10356/86561 http://hdl.handle.net/10220/44074 10.1007/s12257-017-0127-y 202682 en Biotechnology and Bioprocess Engineering © 2017 The Korean Society for Biotechnology and Bioengineering and Springer |
| spellingShingle | Multiple noncanonical amino acids Fluoroproline Deepankumar, Kanagavel Prabhu, Nadarajan Saravanan Kim, June-Hyung Yun, Hyungdon Protein engineering for covalent immobilization and enhanced stability through incorporation of multiple noncanonical amino acids |
| title | Protein engineering for covalent immobilization and enhanced stability through incorporation of multiple noncanonical amino acids |
| title_full | Protein engineering for covalent immobilization and enhanced stability through incorporation of multiple noncanonical amino acids |
| title_fullStr | Protein engineering for covalent immobilization and enhanced stability through incorporation of multiple noncanonical amino acids |
| title_full_unstemmed | Protein engineering for covalent immobilization and enhanced stability through incorporation of multiple noncanonical amino acids |
| title_short | Protein engineering for covalent immobilization and enhanced stability through incorporation of multiple noncanonical amino acids |
| title_sort | protein engineering for covalent immobilization and enhanced stability through incorporation of multiple noncanonical amino acids |
| topic | Multiple noncanonical amino acids Fluoroproline |
| url | https://hdl.handle.net/10356/86561 http://hdl.handle.net/10220/44074 |
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