Production of humanized Fab fragment against human high affinity IgE receptor in Pichia pastoris.
Binding of allergen-IgE complexes to the high affinity IgE receptor (Fc epsilonRI) on mast cells and basophils leads to the release of various mediaters such as histamine. Fab fragments prepared by the papain digestion of humanized antibody against human Fc epsilonRI inhibited the release of histamine from human basophils. Here we established an expression system to directly produce Fab fragments of the humanized anti-human Fc epsilonRI antibody in methylotropic yeast, P. pastoris. Fab fragments were efficiently secreted into the medium at a concentration of 10-40 mg/L using a signal sequence from the P. pastoris phosphatase gene. They were consisted of disulfide-linked light and heavy chains correctly starting from the first amino acid residues by proper cleavage of the signal peptides. The obtained Fab fragments inhibited the binding between IgE and Fc epsilonRI as efficiently as the counterpart prepared by papain digestion of the whole antibody.
Takahashi
K
K
Foods & Pharmaceuticals Research & Development Laboratory, Asahi Breweries Ltd., Kitasoma-gun, Ibaraki, Japan. kyoko.takahashi@asahibeer.co.jp
Yuuki
T
T
Takai
T
T
Ra
C
C
Okumura
K
K
Yokota
T
T
Okumura
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D018076:DNA, Complementary; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D017455:Receptors, IgE / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D018076:DNA, Complementary; D007140:Immunoglobulin Fab Fragments; D017455:Receptors, IgE; D011994:Recombinant Proteins