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Protein engineering for covalent immobilization and enhanced stability through incorporation of multiple noncanonical amino acids

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Abstract

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.

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Authors and Affiliations

  1. School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore city, Singapore

    Kanagavel Deepankumar

  2. Department of Systems Biotechnology, Konkuk University, Seoul, Korea

    Nadarajan Saravanan Prabhu & Hyungdon Yun

  3. Department of Chemical Engineering, College of Engineering, Dong-A University, Busan, Korea

    June-Hyung Kim

Authors
  1. Kanagavel Deepankumar
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  2. Nadarajan Saravanan Prabhu
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  3. June-Hyung Kim
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  4. Hyungdon Yun
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Correspondence to Hyungdon Yun.

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Deepankumar, K., Prabhu, N.S., Kim, JH. et al. Protein engineering for covalent immobilization and enhanced stability through incorporation of multiple noncanonical amino acids. Biotechnol Bioproc E 22, 248–255 (2017). https://doi.org/10.1007/s12257-017-0127-y

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  • DOI: https://doi.org/10.1007/s12257-017-0127-y

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