The assignee for this patent application is
Reporters obtained the following quote from the background information supplied by the inventors: "Membrane receptor proteins located in epithelial cells of insect midguts are target sites for the action of crystalline protein toxins (cry toxins) produced by Bacillus thuringiensis (Bt). Activated cry toxins bind to insect receptor proteins on insect midgut epithelial cells and cause toxicity and death to specific insect pests. The specific interactions between the receptor and toxin are key events in discerning the structure-activity relationships and modes of action of these cry toxins. Being able to measure such interactions is greatly facilitated by the recombinant over production and purification of these receptor proteins to produce sufficient quantities and to reduce the level of non-specific interactions from host impurities. Using purified, functional receptor proteins in biochemical studies allows the measurement of binding affinities and specificities of the receptors in interactions with a range of Bt toxins. However, the membrane receptor proteins are typically hydrophobic, and thus are not soluble in common buffers that are suitable for most protein extractions. A specific purification protocol is described here that maximizes the solubility of hydrophobic insect membrane-bound proteins while preserving their function as receptors to toxins.
"There are many reports of purifying membrane-bound proteins from recombinant hosts using N-laurylsarcosine (N-Dodecyl-N-methylglycine). See, for example, references [1-4]. However, none describes the use of a 1.0% to 0.1% gradient which the inventors found to be surprisingly effective at maximizing the solubility and activity of some hydrophobic membrane-bound receptors found in insect intestinal cell that are produced in recombinant Spodoptera frugiperda (Fall Armyworm) Sf9 insect cells. This method maximally solubilizes membrane receptor proteins for high yield, promotes column binding, and blocks protease cleavage. The purified receptors through this method have high purity and have retained biochemical and biological activity."
In addition to obtaining background information on this patent application, NewsRx editors also obtained the inventors' summary information for this patent application: "A method for purifying a membrane-bound protein from a recombinant insect cell which comprises, suspending the recombinant insect cells in a suitable lysis buffer containing N-laurylsarcosine for an effective period of time, and optionally sonicating, centrifuging the lysate, dialyzing the supernatant against a suitable buffer containing 0.15% N-laurylsarcosine, and isolating the membrane-bound protein using one or more suitable chromatographic methods.
BRIEF DESCRIPTION OF THE DRAWINGS
"FIG. 1(A) is an SDS-PAGE of purified European Corn Borer-cadherin (ECB-cadherin) and (B) European Corn Borer-aminopeptidase 1 (ECB-APN1) produced in Sf9 and purified using the claimed methods. SDS-PAGE of purified membrane receptor proteins shows more than 90% purity (See the arrows, FIGS. 1 A & B). These purified receptor proteins were immobilized onto a Biacore CM4 Sensor.RTM. chip and a CrylAc core toxin trypsin-cleaved CrylAc holotoxin) was flowed over the immobilized receptor proteins. The binding interaction was measured with a Biacore.RTM. 3000 surface plasmon resonance instrument. The binding curves are shown in FIGS. 2 and 3. The dissociation constant Kd for CrylAc binding to the APN-1 purified from ECB larvae was 107 nM, and that for the cadherin protein from ECB larvae was 48 nM. These values are similar to those reported in the literature 
"FIG. 2 is a binding curve of CrylAc core toxin with ECB-APN1.
"FIG. 3 is a binding curve of CrylAc core toxin with ECB-cadherin."
For more information, see this patent application:
Keywords for this news article include: Peptides, Proteins, Chemistry, Amino Acids, Biochemical,
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