Bacterial expression and photoaffinity labeling of a pheromone binding protein.
The first high-level production of a binding-active odorant binding protein is described. The expression cassette polymerase chain reaction was used to generate a DNA fragment encoding the pheromone binding protein (PBP) of the male moth Antheraea polyphemus. Transformation of Escherichia coli cells with a vector containing this construct generated clones which, when induced with isopropyl beta-D-thiogalactopyranoside, produced the 14-kDa PBP in both the soluble fraction and in inclusion bodies. Purification of the soluble recombinant PBP by preparative isoelectric focusing and gel filtration gave > 95% homogeneous protein, which was immunoreactive with an anti-PBP antiserum and exhibited specific, pheromone-displaceable covalent modification by the photoaffinity label [3H]6E,11Z-hexadecadienyl diazoacetate. Recombinant PBP was indistinguishable from the insect-derived PBP, as determined by both native and denaturing gel electrophoresis, immunoreactivity, and photoaffinity labeling properties. Moreover, the insoluble inclusion body protein could be solubilized, refolded, and purified by the same procedures to give a recombinant PBP indistinguishable from the soluble PBP. Proton NMR spectra of the soluble and refolded protein provide further evidence that they possess the same folded structure.
Prestwich
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Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138.
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007301:Insect Hormones / Q000737:chemistry / Q000235:genetics*; D019476:Insect Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008297:Male; D008969:Molecular Sequence Data; D009036:Moths / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D000345:Affinity Labels; D002352:Carrier Proteins; D004274:DNA, Recombinant; D007301:Insect Hormones; D019476:Insect Proteins; D036341:Intercellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; C067715:APR-1 pheromone-binding protein, Antheraea pernyi