VIRGINIAE BUTANOLIDE BINDING-PROTEIN FROM STREPTOMYCES-VIRGINIAE - EVIDENCE THAT VBRA IS NOT THE VIRGINIAE BUTANOLIDE BINDING-PROTEIN AND REIDENTIFICATION OF THE TRUE BINDING-PROTEIN
S. Okamoto et al., VIRGINIAE BUTANOLIDE BINDING-PROTEIN FROM STREPTOMYCES-VIRGINIAE - EVIDENCE THAT VBRA IS NOT THE VIRGINIAE BUTANOLIDE BINDING-PROTEIN AND REIDENTIFICATION OF THE TRUE BINDING-PROTEIN, The Journal of biological chemistry, 270(20), 1995, pp. 12319-12326
Virginiae butanolides (VBs) A-E are butyrolactone autoregulators that
control virginiamycin production in Streptomyces virginiae. We have pr
eviously reported the purification and molecular cloning of VbrA, a pu
tative VB binding protein (Okamoto, S., Nihira, T., Kataoka, H., Suzuk
i, A., and Yamada, Y. (1992) J. Biol. Chem. 267, 1093-1098). However,
VbrA protein overexpressed in Escherichia coli did not show any detect
able VB binding activity nor did the immunoprecipitation of native Vbr
A from a cell-free extract of S. virginiae cause any decrease in such
activity, indicating that VbrA is not the true VB binding protein. Thi
s finding prompted us to seek the true VB binding protein by repurific
ation. After successive purification by anion exchange, gel filtration
, heparin, and hydrophobic interaction chromatography, a 26-kDa protei
n (p26k) was identified as the true VB binding protein. Partial amino
acid sequences of p26k were determined, and the gene (barA) that encod
es this protein was isolated and cloned using degenerate oligonucleoti
de probes. When the barA gene was expressed in Streptomyces lividans a
nd E. coli, strong VB binding activity appeared, demonstrating unambig
uously that the S. virginiae p26k protein is the true VB binding prote
in.