Site-Selective Functionalization of Flagellin by Steric Self-Protection: A Strategy To Facilitate Flagellin as a Self-Adjuvanting Carrier in Conjugate Vaccine

Chi Jiun Peng, Hsiu Ling Chen, Cheng Hsun Chiu, Jim Min Fang*

*Corresponding author for this work

Research output: Contribution to journalJournal Article peer-review

6 Scopus citations

Abstract

Flagellin (FliC) can act as a carrier protein in the preparation of conjugate vaccines to elicit a T-cell-dependent immune response and as an intrinsic adjuvant to activate the toll-like receptor 5 (TLR5) to enhance vaccine potency. To enable the use of FliC as a self-adjuvanting carrier, an effective method for site-selective modification (SSM) of pertinent amino-acid residues in the D2 and D3 domains of FliC is explored without excessive modification of the D0 and D1 domains, which are responsible for activating and binding with TLR5. In highly concentrated Na2SO4 solution, FliC monomers form flagellar filaments, in which the D0 and D1 domains are situated inside the tubular structure. Thus, the lysine residues (K219, K224, K324, and K331) in the D2 and D3 domains of flagellin are selectively modified by a diazo-transfer reaction with imidazole-1-sulfonyl azide. The sites with azido modification are confirmed by MALDI-TOF-MS, ESI-TOF-MS, and LC–MS/MS analyses along with label-free quantitation. The azido-modified filament dissolves to give FliC monomers, which can conjugate with alkyne-hinged saccharides by the click reaction. Transmission electron microscopy imaging, dynamic light scattering measurements, and the secreted embryonic alkaline phosphatase reporter assay indicate that the modified FliC monomers retain the ability either to bind with TLR5 or to reassemble into filaments. Overall, this study establishes a feasible method for the SSM of FliC by steric self-protection of the D0 and D1 domains.

Original languageEnglish
Pages (from-to)805-814
Number of pages10
JournalChemBioChem
Volume19
Issue number8
DOIs
StatePublished - 16 04 2018

Bibliographical note

Publisher Copyright:
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Keywords

  • antigens
  • conjugate vaccines
  • flagellins
  • self-assembly
  • steric hindrance

Fingerprint

Dive into the research topics of 'Site-Selective Functionalization of Flagellin by Steric Self-Protection: A Strategy To Facilitate Flagellin as a Self-Adjuvanting Carrier in Conjugate Vaccine'. Together they form a unique fingerprint.

Cite this