Oleosins contain a unique hydrophobic domain which is inserted into the oil matrix and are involved in the formation and stability of plant oil bodies. These proteins have also been reported to possess some allergenic properties. Therefore, knowledge of its three-dimensional structure is vital for further structural and immunological characterization. However, due to the difficulty of soluble recombinant expression in Escherichia coli, no studies have been done in line with this goal. Here, we have developed a novel expression and purification system for three peanut oleosin isoforms (14 k, 16 k, and 18 kDa oleosins). Oleosin cDNAs were cloned and subsequently expressed in soluble form in insect cell-baculovirus system. Recombinant proteins can be purified to homogeneity using only Ni Sepharose affinity chromatography. Thermal denaturation midpoint temperatures of recombinant oleosins were also assayed and found to be very similar to that of native oleosins, indicating proper structural conformation of the recombinant proteins.

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Using supported lipid monolayers we have studied here calcium signals in antigen-specific B-cells (TNP-specific B-cell hybridomas, TP67.21) triggered by lipid hapten (TNP-Cap-DPPE). Stimulation of the B-cell hybridomas (TP67.21) with a supported DPPC monolayers containing 1% TNP-Cap-DPPE increased the intracellular free calcium ion concentration [Ca2+]i in B-cells. None of B-cells responded to a DPPC monolayers without lipid hapten (TNF-Cap-DPPE). Triggering for calcium signals was clearly dependent on the fluidity of the lipid monolayers. Solid DPPC and DSPC monolayers triggered the calcium signals more efficiently than the fluid DMPC monolayers did. These calcium signals became apparently more efficient in the presence of cholesterol. All of these results suggested that the rigidity of cross-linking for antigen receptors (mIgM) may be a crucial role for triggering calcium signals in B-cells.