Fast large-scale purification of tetracycline repressor variants from overproducing Escherichia coli strains

Ettner, Norbert; Müller, Gerhard; Berens, Christian; Backes, Heike; Schnappinger, Dirk; Schreppel, Thomas; Pfleiderer, Klaus; Hillen, Wolfgang

doi:10.1016/0021-9673(96)00232-4

Cited by 45 publications

(47 citation statements)

References 34 publications

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“…The plasmid pET-3c-yvoA (see Table 2) was constructed for overexpression of YvoA by cloning yvoA amplified with the primers yvoA_ov_fw and yvoA_ov_rev into pET-3c (Novagen) via NdeI/ BamHI. Native YvoA was purified using a protocol and the same conditions established for purification of TetR (19). Briefly, E. coli FT1/pLysS (44) was transformed with pET-3c-yvoA, and T7-polymerase-dependent yvoA transcription was induced by isopropyl-␤-D-thiogalactopyranoside (IPTG).…”

Section: Methodsmentioning

confidence: 99%

Regulon of the N -Acetylglucosamine Utilization Regulator NagR in Bacillus subtilis

et al. 2011

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N-Acetylglucosamine (GlcNAc) is the most abundant carbon-nitrogen biocompound on earth and has been shown to be an important source of nutrients for both catabolic and anabolic purposes in Bacillus species. In this work we show that the GntR family regulator YvoA of Bacillus subtilis serves as a negative transcriptional regulator of GlcNAc catabolism gene expression. YvoA represses transcription by binding a 16-bp sequence upstream of nagP encoding the GlcNAc-specific EIIBC component of the sugar phosphotransferase system involved in GlcNAc transport and phosphorylation, as well as another very similar 16-bp sequence upstream of the nagAB-yvoA locus, wherein nagA codes for N-acetylglucosamine-6-phosphate deacetylase and nagB codes for the glucosamine-6-phosphate (GlcN-6-P) deaminase. In vitro experiments demonstrated that GlcN-6-P acts as an inhibitor of YvoA DNA-binding activity, as occurs for its Streptomyces ortholog, DasR. Interestingly, we observed that the expression of nag genes was still activated upon addition of GlcNAc in a ⌬yvoA mutant background, suggesting the existence of an auxiliary transcriptional control instance. Initial computational prediction of the YvoA regulon showed a distribution of YvoA binding sites limited to nag genes and therefore suggests renaming YvoA to NagR, for N-acetylglucosamine utilization regulator. Whole-transcriptome studies showed significant repercussions of nagR deletion for several major B. subtilis regulators, probably indirectly due to an excess of the crucial molecules acetate, ammonia, and fructose-6-phosphate, resulting from complete hydrolysis of GlcNAc. We discuss a model deduced from NagR-mediated gene expression, which highlights clear connections with pathways for GlcNAc-containing polymer biosynthesis and adaptation to growth under oxygen limitation.

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Section: Methodsmentioning

confidence: 99%

Regulon of the N -Acetylglucosamine Utilization Regulator NagR in Bacillus subtilis

et al. 2011

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“…PWH1919 contains restriction sites for BstXI and MluI (14), pWH620, in addition to the BstXI and MluI sites, also sites for SauI and NcoI (see below). The plasmid pWH1950 used for overexpression of TetR variants has also been described (24). The phage tet50 (20,25) used in the ␤-galactosidase assays and the M13mp19 derivative mWH892 (14) used for oligonucleotide-directed mutagenesis have been described.…”

Section: Methodsmentioning

confidence: 99%

The Role of the Variable Region in Tet Repressor for Inducibility by Tetracycline

Berens

Schnappinger

Hillen

1997

Journal of Biological Chemistry

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“…tetR mutants present in pWH1919 based plasmids [12] were cloned as XbaIϪSphI fragments into pWH1950. Overproduction and purification of TetR and TetR mutants was done as described [8].…”

Section: Methodsmentioning

confidence: 99%

Affinities of mAbs to Tet repressor complexed with operator or tetracycline suggest conformational changes associated with induction

Pook

Grimm

Bonin

et al. 1998

European Journal of Biochemistry

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We isolated five monoclonal antibodies (mAbs) made against tetracycline repressor (TetR), one against the TetR tetracycline complex (Tc) and two against theTetR‐tet operator (tetO) complex. The epitopes of the anti‐TetR mAbs are localized in the α‐helix‐turn‐α‐helix motif (HTH), at different sites near the Tc binding pocket and at the dimerization interface. The anti‐TetR‐Tc and one of the anti‐TetR‐tetO mAbs recognize epitopes near the Tc binding pocket. The other anti‐TetR‐tetO mAb binds to an epitope within the HTH. Quantitative immunoprecipitation and competitive ELISA employing TetR, TetR‐Tc, or TetR‐tetO revealed different affinities of the mAbs for TetR in these functional states. Binding of the two mAbs to epitopes in the HTH was identical for TetR and TetR‐Tc indicating the same conformation in both forms. The epitope located in the dimerization interface is bound more strongly in TetR compared to TetR‐Tc, supporting the idea of different conformations of that epitope in these forms of TetR. The greatest affinity differences were found for epitopes around the Tc binding pocket. Two anti‐TetR mAbs have the highest affinities for free TetR, somewhat reduced affinity for TetR‐tetO and the lowest affinities for TetR‐Tc. The anti‐TetR‐Tc mAb has a discontinuous epitope, formed in TetR‐Tc, which is less well bound in TetR and not bound in the TetR‐tetO complex. One anti‐TetR‐tetO mAb does not recognize TetR‐Tc. Since the epitopes do not overlap with the respective ligand binding sites on TetR, these results are interpreted as conformational differences of the epitopes in these forms of TetR.

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Fast large-scale purification of tetracycline repressor variants from overproducing Escherichia coli strains

Cited by 45 publications

References 34 publications

Regulon of the N -Acetylglucosamine Utilization Regulator NagR in Bacillus subtilis

Regulon of the N -Acetylglucosamine Utilization Regulator NagR in Bacillus subtilis

The Role of the Variable Region in Tet Repressor for Inducibility by Tetracycline

Affinities of mAbs to Tet repressor complexed with operator or tetracycline suggest conformational changes associated with induction

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