Virginiae butanolide binding protein from Streptomyces virginiae. Evidence that VbrA is not the virginiae butanolide binding protein and reidentification of the true binding protein.
Virginiae butanolides (VBs) A-E are butyrolactone autoregulators that control virginiamycin production in Streptomyces virginiae. We have previously reported the purification and molecular cloning of VbrA, a putative VB binding protein (Okamoto, S., Nihira, T., Kataoka, H., Suzuki, A., and Yamada, Y. (1992) J. Biol. Chem. 267, 1093-1098). However, VbrA protein overexpressed in Escherichia coli did not show any detectable VB binding activity nor did the immunoprecipitation of native VbrA from a cell-free extract of S. virginiae cause any decrease in such activity, indicating that VbrA is not the true VB binding protein. This finding prompted us to seek the true VB binding protein by repurification. After successive purification by anion exchange, gel filtration, heparin, and hydrophobic interaction chromatography, a 26-kDa protein (p26k) was identified as the true VB binding protein. Partial amino acid sequences of p26k were determined, and the gene (barA) that encodes this protein was isolated and cloned using degenerate oligonucleotide probes. When the barA gene was expressed in Streptomyces lividans and E. coli, strong VB binding activity appeared, demonstrating unambiguously that the S. virginiae p26k protein is the true VB binding protein.
Okamoto
S
S
Department of Biotechnology, Faculty of Engineering, Osaka University, Japan.
Nakamura
K
K
Nihira
T
T
Yamada
Y
Y
D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors
D001426:Bacterial Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C082569:VbrA protein, Streptomyces virginiae; C093490:barA protein, Streptomyces virginiae; C052546:virginiamycin butanolide C; D015107:4-Butyrolactone