title: Design, production, and characterization of an engineered biotin ligase (BirA) and its application for affinity purification of staphylokinase produced from Bacillus subtilis via secretion.
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.
authors.affiliation: Department of Biological Sciences, Division of Cellular, Molecular, and Microbial Biology, University of Calgary, 2500 University Drive, N.W., Calgary, Alberta T2N 1N4, Canada.
authors.forename: Sau-Ching
authors.initials: SC
authors.lastname: Wu
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Yeung
authors.forename: Peter M
authors.initials: PM
authors.lastname: Hwang
authors.forename: Sui-Lam
authors.initials: SL
authors.lastname: Wong
mesh_terms: D001360:Avidin; D001412:Bacillus subtilis; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000096:biosynthesis / Q000235:genetics*; D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005818:Genetic Engineering; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012097:Repressor Proteins; D014157:Transcription Factors
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D001360:Avidin; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli