Novel system for in vivo biotinylation and its application to crab antimicrobial protein scygonadin.

Biotechnology Letters
Yifeng Li, Rui Sousa

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.

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Jan 10, 2012·Protein Expression and Purification·Yifeng Li, Rui Sousa

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Citations

Jan 15, 2014·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Min ZangJin Yang
Jun 15, 2019·The Journal of Biological Chemistry·Desirée García-Torres, Carol A Fierke
Dec 29, 2020·Antimicrobial Agents and Chemotherapy·Yujie LiuMingqiang Qiao
Apr 10, 2020·Journal of Chromatography. a·Vlad-Stefan RaducanuSamir M Hamdan
Oct 15, 2021·Biomaterials Science·Angeline PelusCédric Y Montanier

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