Site-specific cross-linking of functional proteins by transglutamination

Microbial transglutaminase (MTG), an enzyme that works in posttranslational modification of proteins, has been applied to in vitro preparation of a bi-functional fusion protein from separately obtained recombinant proteins. A quite simple strategy in which a specific peptidyl linker recognizable by...

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Bibliographic Details
Published in:Enzyme and microbial technology 2003-09, Vol.33 (4), p.492-496
Main Authors: Kamiya, Noriho, Takazawa, Takeshi, Tanaka, Tsutomu, Ueda, Hiroshi, Nagamune, Teruyuki
Format: Article
Language:English
Subjects:
Biological and medical sciences
Biotechnology
Enzymatic cross-linking
Enzymatic protein labeling
Fundamental and applied biological sciences. Psychology
Methods. Procedures. Technologies
Multi-functional protein
Peptidyl linker
Protein engineering
Site-specific modification
Transglutaminase
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Summary:Microbial transglutaminase (MTG), an enzyme that works in posttranslational modification of proteins, has been applied to in vitro preparation of a bi-functional fusion protein from separately obtained recombinant proteins. A quite simple strategy in which a specific peptidyl linker recognizable by MTG is fused to the N-terminus of proteins of interest has been verified in the preparation of a bi-functional fusion protein using an antibody variable domain (single chain Fv of anti-hen-egg white lysozyme antibody, scFv) and a fluorescent protein (enhanced yellow fluorescent protein, EYFP). The resultant peptidyl linker-fused proteins were readily cross-linked by MTG to only give the heterodimer (i.e. scFv-EYFP fusion protein), suggesting that the reaction proceeded in highly specific manner. As the fusion protein exhibited sufficient bi-functionality in fluorescence immunoassay (FIA), this work shows for the first time a successful enzymatic preparation of a bi-functional fusion protein through recombinantly incorporated specific peptidyl linkers into target proteins.
ISSN:0141-0229
1879-0909
DOI:10.1016/S0141-0229(03)00154-6