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Effective preparation method of human catalytic antibody light chains
Author(s) -
Uda Taizo,
Honjo Eijiro,
Hifumi Emi
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.928.13
Subject(s) - immunoglobulin light chain , chemistry , catalytic triad , antibody , peptide , catalysis , complementary dna , amidase , substrate (aquarium) , antigen , enzyme , peptide bond , microbiology and biotechnology , biochemistry , active site , biology , gene , ecology , genetics , immunology
We have successfully produced mouse type catalytic antibody light chains (antigenases) by immunizing ground‐state peptides or proteins. The antigenase possesses the ability to enzymatically decompose a targeting molecule using the catalytic triad formed in the structure. In the case of human antibody light chains, the catalytic triad was mostly encoded in Subgroup II, suggesting that the light chains belonging to the subgroup should exhibit enzymatic functions. In order to verify the above concept, we amplified an antibody germline gene as the cDNA belonging to Subgroup II, which was taken from human leucocyte. The cDNA was inserted in pET system, followed by a transformation to express the protein (human antibody light chain) in E.coli . We could recover the protein with very high purity by passing two affinity columns. Its catalytic activity was measured using synthetic substrate (peptide‐MCA) to monitor the fluorescence generated by the cleavage of the peptide‐MCA bond, suggesting the human antibody light chain exhibited the amidase activity. Furthermore, the light chains showed other interesting enzymatic reaction in addition to the amidase activity.