Abstract
Recombinant bovine cardiac sodium–calcium exchange (NCX1) in a baculovirus construct was used to infect cabbage looper larvae (Trichoplusia ni). Infected larvae were homogenized and larvae membrane vesicles were purified. Western blot analysis indicated the presence of recombinant NCX1 protein in vesicles from infected larvae but not in controls. Vesicles from infected larvae expressed high levels of NCX1 activity (1.7 nmol Ca2+/mg protein/s) while vesicles from control larvae had no activity. NCX1 in larvae vesicles was bidirectional. Kinetic analysis yielded aVmaxof 3.6 nmol Ca2+/mg protein/s and aKmfor Ca of 4.2 μM. NCX1 activity was inhibited by the exchange inhibitory peptide with an IC50of 4 μM. These data demonstrate a novel and efficient method for the expression of large amounts of active recombinant NCX1 protein that has general application for expression and analysis of recombinant membrane proteins.
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