title: Novel system for in vivo biotinylation and its application to crab antimicrobial protein scygonadin.
abstract: BirA is a biotin ligase from Escherichia coli that specifically biotinylates a lysine side-chain within a 15-amino acid acceptor peptide (also known as Avi-tag). We developed a protocol for producing recombinant BirA ligase in E. coli for in vitro biotinylation (Li and Sousa, Prot Expr Purif, 82:162-167, 2012) in which the target protein was expressed as both thioredoxin and MBP fusions, and was released by TEV protease-mediated cleavage. The liberated ligase and the fusion proteins were enzymatically active. Based on that observation, we have now developed a novel system for in vivo biotinylation by co-expressing the Avi-tagged target protein with the MBP-BirA fusion. The effectiveness of this system was demonstrated by the successful in vivo labeling of antimicrobial protein, scygonadin. This new system shows improved efficiency compared with pre-existing one and this is likely attributed to the high expression level and solubility of the co-expressed MBP-BirA.
authors.affiliation: Protein Production Core Facility, Department of Biochemistry, University of Texas Health Science Center at San Antonio, Medical Building 431B, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA. liy10@uthscsa.edu
authors.forename: Yifeng
authors.initials: Y
authors.lastname: Li
authors.forename: Rui
authors.initials: R
authors.lastname: Sousa
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000378:metabolism*; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D060847:Metabolic Engineering; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D023181:Antimicrobial Cationic Peptides; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli