Functional expression of pheromone binding protein

Details Functional expression of pheromone binding protein Functional expression of pheromone binding protein

1999-02-24

2001-11-13

Nolan, Patrick J. (Department: 1644)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C530S353000, C530S858000

Reexamination Certificate

active

06316221

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the pheromone binding protein and a method for its functional expression, and more particularly to the recombinant pheromone binding protein (rPBP) of
Bombyx mori
and a method for its expression.
The recombinant pheromone binding protein (rPBP) of
Bombyx mori
is useful not only for studies of it's 3-dimensional structure and ligand binding, but also for ligand-binding protein complex and olfactory processing studies.
BACKGROUND OF THE INVENTION
Odorant binding proteins (OBP) are the most abundant components of the lymph in the antenna of insects.
These proteins are believed to participate in transport, protection and/or inactivation of information in the early events of insect olfaction.
Odorant binding proteins have been studied in Lepidoptera, where these are divided into two groups at least.
The pheromone binding proteins (PBP) belong to one group of the odorant binding proteins and have been distinguished as proteins which participate in recognition of (sex) pheromones.
Further, the proteins which belong to another group of the odorant binding proteins participate in the recognition of general odorants (general odorant binding proteins, GOBP).
The pheromone binding proteins which participate in recognition of sex pheromones have been detected and characterized in a number of species from several insects orders since the first identification of these proteins in
Antheraea pilophemus.
Functionally, these proteins can be classified as lipocalins, a group of proteins binding hydrophobic ligands.
However, insect odor molecular binding proteins (insect OBP) share little homology with other lipocalins, which are primarily composed of 8 or 10 antiparallel &bgr;-strands forming &bgr;-barrel.
Circular dichroism measurement and theoretical structure calculation revealed that insect OBPs are in a large part &agr;-helical.
Therefore, the study of 3-dimensional structure and that of ligand binding of insect OBPs enable to get a better understanding of the role of these proteins in the olfactory Processing.
Though reasonable Amount of functional OBP has been desired for the research in various fields, the isolation of sufficient amount of nature?? protein is out of the question.
Insect odor molecular binding proteins have been previously expressed both in bacterial and eukaryotic systems.
Antheraea pernyi
has been expressed in the baculovirus system, but it's not a perfect method for the production of sufficient amount of insect OBP because of low yield.
While the PBP from
Antheraea polyphemus
has been expressed in low yield in
Escherichia coli
, the product is physiologically inactivated and required refolding for structural and functional studies.
Therefore, a method which enable to provide sufficient amount of insect OBP, furthermore, to overexpress under the condition of physiological activation has been in demand.
SUMMARY OF THE INVENTION
We studied said problem and found that the functional recombinant pheromone binding protein from
Bombyx mori
is successfully obtained in high yield in
Escherichia coli
by pET-22b vector which contains the pelB signal peptide, moreover, better expression efficiency is achieved at 29° C. rather than at a usual temperature (37° C.), and in the absence of the inducing agent (IPTG).
In short, means for attaining the object of invention are as follows:
(1) The functional pheromone binding protein from
Bombyx mori
, whereby it is expressed with
Escherichia coli
by the pET-22b vector which contains the N-terminal pelB signal peptide.
(2) A method for functional expression of pheromone binding protein from
Bombyx mori
, whereby it is expressed with
Escherichia coli
by the pET-22b vector which contains the N-terminal pelB signal peptide.
(3) A method for functional expression of the pheromone binding protein from
Bombyx mori
, whereby it is expressed with
Escherichia coli
by the pET-22b vector which contains the N-terminal pelB signal peptide, in between 36-38° C., further preferably in between 28-30° C.
(4) A method for functional expression of pheromone binding protein from
Bombyx mori
, whereby it is expressed with
Escherichia coli
by the pET-22b vector which contains the N-terminal pelB signal peptide in between 36-38° C., further preferably in between 28-30° C., and in the absence of the inducing agent (IPTG).
The recombinant protein of the present invention efficiently bound to the radiolabeled pheromone (bombykol) in native gel electrophoresis assay.
According to the present invention, it is possible to express sufficient amount of insect OBP, moreover the production under the condition of physiological activation is also enabled.

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