Nucleic Acids Research, Vol 26, Issue 6 1414-1420, Copyright © 1998 by Oxford University Press
PA Smith, BC Tripp, EA DiBlasio-Smith, Z Lu, ER LaVallie and JM McCoy
The high affinity binding interaction of biotin to avidin or streptavidin
has been used widely in biochemistry and molecular biology, often in
sensitive protein detection or protein capture applications. However, in
vitro chemical techniques for protein biotinylation are not always
successful, with some common problems being a lack of reaction specificity,
inactivation of amino acid residues critical for protein function and low
levels of biotin incorporation. This report describes an improved
expression system for the highly specific and quantitative in vivo
biotinylation of fusion proteins. A short 'biotinylation peptide',
described previously by Schatz, is linked to the N-terminus of Escherichia
coli thioredoxin (TrxA) to form a new protein, called BIOTRX. The
'biotinylation peptide' serves as an in vivo substrate mimic for E. coli
biotin holoenzyme synthetase (BirA), an enzyme which usually performs
highly selective biotinylation of E.coli biotin carboxyl carrier protein
(BCCP). A plasmid expression vector carrying the BIOTRX and birA genes
arranged as a bacterial operon can be used to obtain high level production
of soluble BIOTRX and BirA proteins and, under appropriate culture
conditions, BIOTRX protein produced by this system is completely
biotinylated. Fusions of BIOTRX to other proteins or peptides, whether
these polypeptides are linked to the C-terminus or inserted into the BIOTRX
active site loop, are also quantitatively biotinylated. Both types of
BIOTRX fusion can be captured efficiently on avidin/streptavidin media for
purification purposes or to facilitate interaction assays. We illustrate
the utility of the system by measurements of antibody and soluble receptor
protein binding to BIOTRX fusions immobilized on streptavidin-conjugated
BIAcore chips.
ARTICLES
A plasmid expression system for quantitative in vivo biotinylation of thioredoxin fusion proteins in Escherichia coli
Genetics Institute Inc., 87 Cambridge Park Drive, Cambridge, MA 02140, USA.
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