首页 | 本学科首页   官方微博 | 高级检索  
   检索      


Design, production, and characterization of an engineered biotin ligase (BirA) and its application for affinity purification of staphylokinase produced from Bacillus subtilis via secretion
Authors:Wu Sau-Ching  Yeung Jonathan C  Hwang Peter M  Wong Sui-Lam
Institution:Department of Biological Sciences, Division of Cellular, Molecular, and Microbial Biology, University of Calgary, 2500 University Drive, N.W., Calgary, Alberta T2N 1N4, Canada.
Abstract:A major attraction in using Bacillus subtilis as an expression host for heterologous protein production is its ability to secrete extracellular proteins into the culture medium. To take full advantage of this system, an efficient method for recovering the target protein is crucial. For secretory proteins which cannot be purified by a simple scheme, in vitro biotinylation using biotin ligase (BirA) offers an effective alternative for their purification. The availability of large amounts of quality BirA can be critical for in vitro biotinylation. We report here the engineering and production of an Escherichia coli BirA and its application in the purification of staphylokinase, a fibrin-specific plasminogen activator, from the culture supernatant of Bacillus subtilis via in vitro biotinylation. BirA was tagged with both a chitin-binding domain and a hexahistidine tail to facilitate both its purification and its removal from the biotinylated sample. We show in this paper how, in a unique way, we solved the problem of protein aggregation in the E. coli BirA production system to achieve a yield of soluble functional BirA hitherto unreported in the literature. Application of this novel BirA to protein purification via in vitro biotinylation in general will also be discussed. Biotinylated staphylokinase produced in the study not only can act as an intermediate for easy purification, it can also serve as an important element in the creation of a blood clot targeting and dissolving agent.
Keywords:
本文献已被 ScienceDirect PubMed 等数据库收录!
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号