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A Plasmid Expression System For Quantitative In Vivo Biotinylation Of Thioredoxin Fusion Proteins In Escherichia Coli.

P. A. Smith, B. Tripp, E. DiBlasio-Smith, Z. Lu, E. Lavallie, J. Mccoy
Published 1998 · Biology, Medicine

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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.
This paper references
10.1042/BJ1010774
Thermodynamics of the binding of biotin and some analogues by avidin.
N. Green (1966)
Biochem. J
N M Green (1966)
Adv. Protein Chem
N M Green (1975)
10.1016/0003-2697(79)90374-9
An avidin monomer affinity column for the purification of biotin-containing enzymes.
K. Henrikson (1979)
10.1016/0076-6879(79)62246-2
[66] Analysis of microbial biotin proteins
R. Fall (1979)
Anal. Biochem
K P Henrikson (1979)
Methods Enzymol
R R Fall (1979)
The use of the 2-iminobiotin-avidin interaction for the selective retrieval of labeled plasma membrane components.
G. Orr (1981)
10.1016/0022-2836(81)90043-7
Genetic and biochemical characterization of the birA gene and its product: evidence for a direct role of biotin holoenzyme synthetase in repression of the biotin operon in Escherichia coli.
D. Barker (1981)
10.1016/0022-2836(81)90042-5
The birA gene of Escherichia coli encodes a biotin holoenzyme synthetase.
D. Barker (1981)
Current Protocols in Molecular Biology, Unit 16
J M Mccoy (1981)
J. Mol. Biol
Barker (1981)
J. Mol. Biol
D F Barker (1981)
Anal. Biochem
G Heney (1981)
Molecular Cloning: A Laboratory Manual
J. Sambrook (1983)
10.1016/0378-1119(85)90011-3
Nucleotide sequence of the birA gene encoding the biotin operon repressor and biotin holoenzyme synthetase functions of Escherichia coli.
P. K. Howard (1985)
10.1016/0003-2697(81)90456-5
Purification of avidin and its derivatives on 2-iminobiotin-6-aminohexyl-Sepharose 4B.
G. Orr (1986)
10.1016/s0021-9258(18)68661-2
Evolutionary conservation among biotin enzymes.
D. Samols (1988)
J. Biol. Chem
D Samols (1988)
Molecular Cloning: A Laboratory Manual, 2nd Edn
J Sambrook (1989)
10.1016/0022-2836(90)90313-B
Crystal structure of thioredoxin from Escherichia coli at 1.68 A resolution.
S. K. Katti (1990)
10.1016/0076-6879(90)84268-L
[14] Protein biotinylation
E. Bayer (1990)
10.1016/0076-6879(90)84256-G
Introduction to avidin-biotin technology.
M. Wilchek (1990)
10.1016/s0021-9258(18)86949-6
Biotination of proteins in vivo. A post-translational modification to label, purify, and study proteins.
J. Cronan (1990)
J. Biol. Chem
J E Cronan (1990)
Methods Enzymol
E A Bayer (1990)
Methods Enzymol
M Wilchek (1990)
10.1271/BBB.56.1017
In vivo biotinylation of fusion proteins expressed in Escherichia coli with a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit.
N. Yamano (1992)
Biosci. Biotechnol. Biochem
N Yamano (1992)
10.1073/PNAS.90.11.5076
Three-dimensional structures of avidin and the avidin-biotin complex.
O. Livnah (1993)
10.1038/NBT1093-1138
Use of Peptide Libraries to Map the Substrate Specificity of a Peptide-Modifying Enzyme: A 13 Residue Consensus Peptide Specifies Biotinylation in Escherichia coli
P. Schatz (1993)
10.1038/NBT0293-187
A Thioredoxin Gene Fusion Expression System That Circumvents Inclusion Body Formation in the E. coli Cytoplasm
Edward R. LaVallie (1993)
Proc. Natl. Acad. Sci. USA, 90
O Livnah (1993)
10.1021/JA00148A003
Molecular Origins of the Slow Streptavidin-Biotin Dissociation Kinetics
A. Chilkoti (1995)
10.1038/NBT0495-366
Expression of Thioredoxin Random Peptide Libraries on the Escherichia coli Cell Surface as Functional Fusions to Flagellin: A System Designed for Exploring Protein-Protein Interactions
Z. Lu (1995)
J. Am. Chem Soc
A Chilkoti (1995)
10.1074/JBC.271.9.5059
Histidine Patch Thioredoxins
Z. Lu (1996)
10.1038/380548A0
Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2
P. Colas (1996)
10.1016/0378-1119(95)00762-8
A versatile plasmid expression vector for the production of biotinylated proteins by site-specific, enzymatic modification in Escherichia coli.
K. L. Tsao (1996)
J. Biol. Chem
Z Lu (1996)



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10.3389/fgene.2020.00450
Proximity Labeling Techniques to Study Chromatin
Henning Ummethum (2020)
10.1074/jbc.RA120.012783
Mammalian lectin arrays for screening host–microbe interactions
Sabine A. F. Jégouzo (2020)
10.1074/jbc.RA118.004101
Conformational heterogeneity of the allosteric drug and metabolite (ADaM) site in AMP-activated protein kinase (AMPK)
X. Gu (2018)
10.1371/journal.pone.0191315
Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection
V. Verma (2018)
Antigen 43-mediated biotin display and fabrication of bacteria-driven microswimmers
Oliver Schauer (2018)
10.1016/b978-0-12-803581-8.10209-7
2.2 Protein-Engineered Biomaterials: Synthesis and Characterization☆
W. Mulyasasmita (2017)
10.1007/978-1-4939-6412-3
Protein Chromatography
D. Walls (2017)
10.1007/978-1-4939-6412-3_8
Tagging Recombinant Proteins to Enhance Solubility and Aid Purification.
Sinéad T. Loughran (2017)
10.1002/9783527683451.CH7
Bioorthogonal Labeling of Cellular Proteins by Enzymatic and Related Mechanisms
Scott A. Walper (2017)
10.1007/978-1-4939-3308-2_14
Preparation of Thermal-Stable Biotinylated Firefly Luciferase and Its Application in Pyrosequencing
Shu-hui Zhu (2016)
10.1002/bit.25810
Clarification technologies for monoclonal antibody manufacturing processes: Current state and future perspectives
Nripen Singh (2016)
Enzymatic Degradation of Organophosphate Compounds: Evaluation of High-level Production, Solubility and Stability
Gholamreza Farnoosh (2016)
Molecular insights into a putative potyvirus RNA encapsidation pathway and potyvirus particles as enzyme nano-carriers
Jane Besong-Ndika (2016)
10.1021/acs.jproteome.5b00142
Upregulation of glycans containing 3' fucose in a subset of pancreatic cancers uncovered using fusion-tagged lectins.
Sudhir Singh (2015)
10.1016/j.ab.2014.09.007
A universal homogeneous assay for high-throughput determination of binding kinetics.
F. Schiele (2015)
10.1038/cr.2014.150
Structural basis of AMPK regulation by adenine nucleotides and glycogen
Xiaodan Li (2015)
10.1126/sciadv.1500107
Structural basis for recognition of diverse transcriptional repressors by the TOPLESS family of corepressors
J. Ke (2015)
10.1016/j.jmb.2015.06.018
A New Versatile Immobilization Tag Based on the Ultra High Affinity and Reversibility of the Calmodulin-Calmodulin Binding Peptide Interaction.
S. Mukherjee (2015)
10.1038/nsmb.2916
Internal motions prime cIAP1 for rapid activation
Aaron H. Phillips (2014)
10.1016/J.BPJ.2013.11.1490
Internal motions prime cIAP1 for rapid activation
A. H. Phillips (2014)
10.1021/jm4009357
Fragment-based identification of a locus in the Sec7 domain of Arno for the design of protein-protein interaction inhibitors.
J. Rouhana (2013)
Structure and function of Norrin in assembly and activation of a Frizzled 4 – Lrp 5 / 6 complex
J. Ke (2013)
10.1101/gad.228544.113
Structure and function of Norrin in assembly and activation of a Frizzled 4-Lrp5/6 complex.
Jiyuan Ke (2013)
Investigation of Hsf1 Interacting Partners via a Genome-wide Yeast Two-hybrid Screen
J. E. Mendez (2013)
10.1073/pnas.1307235110
Rickettsia Sca2 has evolved formin-like activity through a different molecular mechanism
Y. Madasu (2013)
10.1074/jbc.M112.391748
Kinetics of Interaction between ADP-ribosylation Factor-1 (Arf1) and the Sec7 Domain of Arno Guanine Nucleotide Exchange Factor, Modulation by Allosteric Factors, and the Uncompetitive Inhibitor Brefeldin A
J. Rouhana (2012)
Modification of Behavior of Elastin-like Polypeptides by Changing Molecular Architecture
A. Ghoorchian (2012)
10.1016/j.jmb.2012.06.036
Assembling of AcrB trimer in cell membrane.
W. Lu (2012)
10.1007/s10529-012-0942-3
Novel system for in vivo biotinylation and its application to crab antimicrobial protein scygonadin
Yifeng Li (2012)
10.1038/nsmb.2109
Mechanism of actin filament nucleation by Vibrio VopL and implications for tandem-W domain nucleation
S. Namgoong (2011)
10.1074/jbc.M111.244772
Identification of Neutrophil Granule Glycoproteins as Lewisx-containing Ligands Cleared by the Scavenger Receptor C-type Lectin*
Sarah A. Graham (2011)
10.1002/9780470027318.A0206.PUB2
Fluorescence-based Biosensors
M. Schäferling (2011)
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