High-level expression of human CH2domain from the Fc region in Pichia pastoris and preparation of anti-CH2antibodies

Kosuke Oyama, Takatoshi Ohkuri, Mao Inoue, Jose M.M. Caaveiro, Tadashi Ueda

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

Abstract

Pichia pastoris is a popular eukaryotic system employed for the fast, simple and inexpensive production of recombinant protein including biotherapeutics such as human albumin. The CH2 domain of human Immunoglobulin G (IgG) is a promising scaffold for developing novel therapeutics. To accelerate the research of CH2 domain, we have established a procedure to highly express human CH2 domain (∼150 mg/l) as well as human Fc (∼30 mg/l) in yeast P. pastoris. The procedure yields, simultaneously, a major glycosylated (∼70%) and non-glycosylated (∼30%) fractions. They can be easily separated with high purity. Although both forms of CH2 domain have essentially the same secondary structure, the presence of the glycan increased the thermal stability of the CH2 domain by about 5°C as determined from calorimetry. The purified glycosylated CH2 domain elicited polyclonal antibodies in mouse, recognizing not only the CH2 domain, but also recombinant human Fc and the commercial IgG1 antibody Rituxan. Protein A and Protein G binding to the kink region between CH2 domain and CH3 domain of human Fc are used to purify therapeutic proteins. Therefore, these antibodies are candidates to develop a novel affinity material to purify human antibodies using their CH2 domain.

Original languageEnglish
Pages (from-to)289-297
Number of pages9
JournalJournal of biochemistry
Volume170
Issue number2
DOIs
Publication statusPublished - Aug 1 2021

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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