生物素化
亲和素
生物素
生物化学
肽
融合蛋白
化学
体内
体外
生物
靶蛋白
链霉亲和素
分子生物学
重组DNA
基因
生物技术
作者
M G Cull,Peter J. Schatz
出处
期刊:Methods in Enzymology
日期:2000-01-01
卷期号:: 430-440
被引量:219
标识
DOI:10.1016/s0076-6879(00)26068-0
摘要
This chapter discusses the biotinylation of proteins in vivo and in vitro using small peptide tags. Proteins containing the AviTag can be efficiently biotinylated in vitro using the BirA enzyme from E. coli. This option is desirable when the expression of soluble target protein in E. coli is inefficient, as in the case of proteins that form inclusion bodies when expressed in bacteria or when using an expression system not yet developed for efficient biotinylation, e.g., in yeast or in mammalian cells. Whereas the in vitro reaction can be performed in a complex mixture of proteins, a more desirable option is to use in vitro biotinylation on purified proteins to minimize protease contamination and concentrate the substrate to achieve a more efficient reaction. Because the protein may be biotinylated poorly or not at all during expression, a protein purification scheme that does not depend on biotinylated protein is required. Most AviTag fusion proteins can also be efficiently biotinylated in vivo, saving time and eliminating the need for BirA enzyme. Biotinylated proteins can be purified efficiently by exploiting the high degree of specificity between avidin and biotin through the use of monomeric avidin columns. Regardless of the expression vector and host used, efficient in vivo biotinylation of protein- AviTag fusions requires overexpression of BirA.
科研通智能强力驱动
Strongly Powered by AbleSci AI