Preparation and characterization of active site protected poly(ethylene glycol)-avidin bioconjugates

Stefano Salmaso, Alessandra Semenzato, Sara Bersania, Marco Chinol, Giovanni Paganelli, Paolo Caliceti

Research output: Contribution to journalArticle

Abstract

Avidin was modified with poly(ethylene glycol) in the presence of a biotin binding site protective agent synthesised by imminobiotin conjugation to branched 20 kDa PEG. Avidin was incubated with imminobiotin-PEG and reacted with high amounts of 5, 10 or 20 kDa PEG to modify the protein amino groups. Circular dichroism demonstrated that the extensive PEGylation does not alter the protein conformational structure. The affinity of avidin-PEG conjugates for biotin and biotinylated antibodies depended on the PEG size or the use of a protective agent. Avidin-PEG 10 and 20 kDa prepared in the presence of imminobiotin-PEG maintained 100% of the native affinity for biotin. The 5 kDa PEG derivative and the ones obtained without biotin site protection maintained 79-85% of the native affinity. The affinity for biotinylated antibodies decreased to 35% when the conjugation was performed without imminobiotin-PEG, while the conjugates obtained with high-molecular-weight PEGs in the presence of protective agent displayed high residual affinity. All conjugates possessed negligible antigenicity and immunogenicity. PEGylation greatly prolonged the avidin permanence in the circulation, reduced its disposition in the liver and kidneys and promoted accumulation into solid tumors. PEGylation was found to prevent the protein cell uptake, either by phagocytosis or pinocytosis.

Original languageEnglish
Pages (from-to)57-66
Number of pages10
JournalBBA - General Subjects
Volume1726
Issue number1
DOIs
Publication statusPublished - Oct 30 2005

Keywords

  • Active site protection
  • Avidin
  • Avidin/biotin interaction
  • Bioconjugation
  • PEGylation
  • Poly(ethylene glycol)

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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