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Methods Mol Biol. 2017;1586:337-344. doi: 10.1007/978-1-4939-6887-9_22.

In Vivo Biotinylation of Antigens in E. coli.

Author information

1
Structural Genomics Consortium, Department of Biochemistry and Biophysics, Karolinska Institutet, Tomtebodavägen 23a, Gamma:6, 171 65, Solna, Sweden. susanne.graslund@ki.se.
2
Target Discovery Institute and Structural Genomics Consortium, Oxford University, Oxford, UK.
3
Goethe-University Frankfurt, Buchmann Institute for life Sciences, Riedberg Campus, 60438, Frankfurt am Main, Germany.

Abstract

Site-specific biotinylation of proteins is often the method of choice to enable efficient immobilization of a protein on a surface without interfering with protein folding. The tight interaction of biotin and streptavidin is frequently used to immobilize an antigen during phage display selections of binders. Here we describe a method of in vivo biotinylation of proteins during expression in E. coli, by tagging the protein with the short biotin acceptor peptide sequence, Avi tag, and co-expression of the E. coli biotin ligase (BirA) resulting in precise biotinylation of a specific lysine residue in the tag.

KEYWORDS:

Antigen capturing; Antigen immobilization; Avi-tag; Biotinylation; BirA; IMAC; SEC; Streptavidin

PMID:
28470616
DOI:
10.1007/978-1-4939-6887-9_22
[Indexed for MEDLINE]

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