Production of Nα-acetyl Tα1-HSA through in vitro acetylation by RimJ
Thymosin alpha 1 (Tα1) is an important immunomodulating agent with various clinical applications. The natural form of Tα1 is Nα -acetylated, which was supposed to be related to in vivo stability of the hormone. In this study, fusion protein Tα1-HSA was constructed and expressed in Pichia pastoris. RimJ, a Nα -acetyltransferase from E.coli, was also overexpressed and purified to homogeneity. In vitro acetylation of Tα1-HSA in the presence of RimJ and acetyl coenzyme A resulted in Nα -acetyl Tα1-HSA. The Nα -acetylation was determined by LC-MS/MS. Kinetic assay indicated that RimJ had a higher affinity to desacetyl Tα1 than to Tα1-HSA. Bioactivity assay revealed fully retained activity of Tα1 when the hormone was connected to the N-terminus of the fusion protein, while the activity was compromised in our previously constructed HSA-Tα1. With fully retained activity and N-terminal acetylation, Nα -acetyl Tα1-HSA was expected to be a more promising pharmaceutical agent than Tα1.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2017 |
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| Erschienen: |
2017 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:8 |
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| Enthalten in: |
Oncotarget - 8(2017), 56 vom: 10. Nov., Seite 95247-95255 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Chen, Jing [VerfasserIn] |
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| Links: |
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| Themen: |
Acetylation |
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| Anmerkungen: |
Date Revised 20.11.2019 published: Electronic-eCollection Citation Status PubMed-not-MEDLINE |
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| doi: |
10.18632/oncotarget.20259 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM278860575 |
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| 520 | |a Thymosin alpha 1 (Tα1) is an important immunomodulating agent with various clinical applications. The natural form of Tα1 is Nα -acetylated, which was supposed to be related to in vivo stability of the hormone. In this study, fusion protein Tα1-HSA was constructed and expressed in Pichia pastoris. RimJ, a Nα -acetyltransferase from E.coli, was also overexpressed and purified to homogeneity. In vitro acetylation of Tα1-HSA in the presence of RimJ and acetyl coenzyme A resulted in Nα -acetyl Tα1-HSA. The Nα -acetylation was determined by LC-MS/MS. Kinetic assay indicated that RimJ had a higher affinity to desacetyl Tα1 than to Tα1-HSA. Bioactivity assay revealed fully retained activity of Tα1 when the hormone was connected to the N-terminus of the fusion protein, while the activity was compromised in our previously constructed HSA-Tα1. With fully retained activity and N-terminal acetylation, Nα -acetyl Tα1-HSA was expected to be a more promising pharmaceutical agent than Tα1 | ||
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