Metabolic biotinylation of recombinant antibody by biotin ligase retained in the endoplasmic reticulum

被引:41
作者
Barat, Bhaswati [1 ]
Wu, Anna M. [1 ]
机构
[1] Univ Calif Los Angeles, David Geffen Sch Med, Dept Mol & Med Pharmacol, Crump Inst Mol Imaging, Los Angeles, CA 90095 USA
来源
BIOMOLECULAR ENGINEERING | 2007年 / 24卷 / 03期
关键词
biotinylation; carcinoembryonic antigen; engineered antibodies; diabody; biotin ligase; BirA;
D O I
10.1016/j.bioeng.2007.02.003
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Due to its strength and specificity, the interaction between avidin and biotin has been used in a variety of scientific and medical applications ranging from immumohistochemistry to drug targeting. The present study describes two methods for biotinylation of proteins secreted from eukaryotic cells using the Escherichia coli biotin protein ligase. In one system the biotin ligase was co-secreted from the cells along with substrate protein enabling extracellular biotinylation of the tagged protein. In the other system, biotin ligase was engineered to be retained in the endoplasmic reticulum (ER) and metabolically biotinylates the secretory protein as it passes through the ER. An engineered antibody fragment, a diabody with specificity for carcinoembryonic antigen (CEA) was fused to the biotin acceptor domain (123 amino acid) of Propionibacterium shermanii. Coexpression of the fusion protein with ER retained biotin ligase showed higher biotinylation efficiency than biotinylation by co-secreted ligase. Biotinylation of the anti-CEA diabody tagged with a short (15 amino acid, Biotin Avitag (TM)) biotin acceptor peptide was also successful. Utilization of ER retained biotin ligase for biotinylation of protein is an attractive alternative for efficiently producing uniformly biotinylated recombinant proteins for a variety of avidin-biotin technologies. (C) 2007 Elsevier B.V. All rights reserved.
引用
收藏
页码:283 / 291
页数:9
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