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Mammalian expression vectors for metabolic biotinylation tandem affinity tagging by co-expression in cis of a mammalian codon-optimized BirA biotin ligase.

ABSTRACT: ?BJECTIVE: To construct mammalian expression vectors for the N- or C-terminal tagging of proteins with a tandem affinity tag comprised of the biotinylatable Avi tag and of a triple FLAG tag. RESULTS:We constructed and tested by transient transfections mammalian expression vectors for the co-expression from a single plasmid of N- or C-terminally tagged proteins bearing a tandem affinity tag comprised of the biotinylatable Avi tag and of a triple FLAG tag separated by a tobacco etch virus (TEV) protease cleavage site, together with a mammalian codon-optimized BirA biotin ligase fused to green fluorescent protein. We also describe platform vectors for the N- or C-terminal AVI-TEV-FLAG tagging of any complementary DNA of choice. These vectors offer versatility and efficiency in the application of metabolic biotinylation tandem affinity tagging of nuclear proteins in mammalian cells.

SUBMITTER: Ioannou M

PROVIDER: S-EPMC6001059 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Mammalian expression vectors for metabolic biotinylation tandem affinity tagging by co-expression in cis of a mammalian codon-optimized BirA biotin ligase.

Ioannou Marina M Papageorgiou Dimitris N DN Ogryzko Vasily V Strouboulis John J

BMC research notes 20180614 1

ΟBJECTIVE: To construct mammalian expression vectors for the N- or C-terminal tagging of proteins with a tandem affinity tag comprised of the biotinylatable Avi tag and of a triple FLAG tag.<h4>Results</h4>We constructed and tested by transient transfections mammalian expression vectors for the co-expression from a single plasmid of N- or C-terminally tagged proteins bearing a tandem affinity tag comprised of the biotinylatable Avi tag and of a triple FLAG tag separated by a tobacco etch virus ( ...[more]

PMID: 29898783

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Project description:Transcription of the Escherichia coli biotin (bio) operon is directly regulated by the biotin protein ligase BirA, the enzyme that covalently attaches biotin to its cognate acceptor proteins. Binding of BirA to the bio operator requires dimerization of the protein, which is triggered by BirA-catalyzed synthesis of biotinoyl-adenylate (biotinoyl-5'-AMP), the obligatory intermediate of the ligation reaction. Although several aspects of this regulatory system are well understood, no BirA superrepressor mutant strains had been isolated. Such superrepressor BirA proteins would repress the biotin operon transcription in vivo at biotin concentrations well below those needed for repression by wild-type BirA. We isolated mutant strains having this phenotype by a combined selection-screening approach and resolved multiple mutations to give several birA superrepressor alleles, each having a single mutation, all of which showed repression dominant over that of the wild-type allele. All of these mutant strains repressed bio operon transcription in vivo at biotin concentrations that gave derepression of the wild-type strain and retained sufficient ligation activity for growth when overexpressed. All of the strains except that encoding G154D BirA showed derepression of bio operon transcription upon overproduction of a biotin-accepting protein. In BirA, G154D was a lethal mutation in single copy, and the purified protein was unable to transfer biotin from enzyme-bound biotinoyl-adenylate either to the natural acceptor protein or to a biotin-accepting peptide sequence. Consistent with the transcriptional repression data, each of the purified mutant proteins showed increased affinity for the biotin operator DNA in electrophoretic mobility shift assays. Surprisingly, although most of the mutations were located in the catalytic domain, all of those tested, except G154D BirA, had normal ligase activity. Most of the mutations that gave superrepressor phenotypes altered residues located close to the dimerization interface of BirA. However, two mutations were located at sites well removed from the interface. The properties of the superrepressor mutants strengthen and extend other data indicating that BirA function entails extensive interactions among the three domains of the protein and show that normal ligase activity does not ensure normal DNA binding.

Dataset Information

Mammalian expression vectors for metabolic biotinylation tandem affinity tagging by co-expression in cis of a mammalian codon-optimized BirA biotin ligase.

Publications

Mammalian expression vectors for metabolic biotinylation tandem affinity tagging by co-expression in cis of a mammalian codon-optimized BirA biotin ligase.

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