Characterization of deamidation at Asn138 in L-chain of recombinant humanized Fab expressed from Pichia pastoris

Takatoshi Ohkuri, Eri Murase, Shu Lan Sun, Jun Sugitani, Tadashi Ueda

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4 Citations (Scopus)

Abstract

A method was previously established for evaluating Asn deamidation by matrix-assisted laser desorption/ionization time of flight-mass spectrometry using endoproteinase Asp-N. In this study, we demonstrated that this method could be applied to the identification of the deamidation site of the humanized fragment antigen-binding (Fab). First, a system for expressing humanized Fab from methylotrophic yeast Pichia pastoris was constructed, resulting in the preparation of ∼30 mg of the purified humanized Fab from 1 l culture. Analysis of the L-chain derived from recombinant humanized Fab that was heated at pH 7 and 100°C for 1 h showed the deamidation at Asn138 in the constant region. Then, we prepared L-N138D Fab and L-N138A Fab and examined their properties. The circular dichroism (CD) spectrum of the L-N138D Fab was partially different from that of the wild-type Fab. The measurement of the thermostability showed that L-N138D caused a significant decrease in the thermostability of Fab. On the other hand, the CD spectrum and thermostability of L-N138A Fab showed the same behaviour as the wild-type Fab. Thus, it was suggested that the introduction of a negative charge at position 138 in the L-chain by the deamidation significantly affected the stability of humanized Fab.

Original languageEnglish
Pages (from-to)333-340
Number of pages8
JournalJournal of biochemistry
Volume154
Issue number4
DOIs
Publication statusPublished - Oct 1 2013

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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