BIOTIN LIGASE FOR SITE SPECIFIC BIOTINYLATION OF PROTEINS AND PEPTIDES IMMOBILIZED ON MICROPARTICLES
Abstract
To obtain catalytically active recombinant biotin ligase (rBirA), we developed an approach for heterologous expression of BirA-MBP-fusion protein in E. coli cells, its purification and subsequent rBirA release by highly specific proteolysis using TEV protease. The recombinant protein is obtained in homogeneous state and retains specific catalytic activity. Enzyme immobilization on magnetic microparticles has allowed to develop an approach for effective enzymatic biotinylation combined with purification of target protein molecules on the affinity matrix.
Keywords
BirA biotin ligase,
MBP protein,
molecular cloning,
heterologous expression,
site-specific biotinylation
Supporting agencies: This work was partially supported by the Belarussian Foundation for Fundamental Research (grant Х16Р-062 and Х17М-012).
About the Authors
A. V. Svirid
Institute of Bioorganic Chemistry of the National Academy of Sciences of Belarus, Minsk
Belarus
Belarus
Researcher
5/2, Acad. Kuprevich Str., 220141
A. Ya. Lushchyk
Institute of Bioorganic Chemistry of the National Academy of Sciences of Belarus, Minsk
Belarus
Belarus
Researcher
5/2, Acad. Kuprevich Str., 220141
A. A. Gilep
Institute of Bioorganic Chemistry of the National Academy of Sciences of Belarus, Minsk
Belarus
Belarus
Ph. D. (Chemistry), Head of the Department
5/2, Acad. Kuprevich Str., 220141
S. A. Usanov
Institute of Bioorganic Chemistry of the National Academy of Sciences of Belarus, Minsk
Belarus
Belarus
Corresponding Member of the National Academy of Sciences of Belarus, D. Sc. (Chemistry), Professor
5/2, Acad. Kuprevich Str., 220141
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