[Show abstract][Hide abstract] ABSTRACT:
We recently identified a region located in the extramembrane loop 13(L13) of SGLT1 critically involved in phlorizin binding. To investigate the structure-function relationship, large amounts of L13 are needed. Here we describe a high level expression using the RTS 500 in vitro translation system. A gene coding for L13 was subcloned into an in vitro translation vector, pIVEX. Under the control of T7 promoter, L13 could be high level expressed in vitro. The amount of L13 could reach up to 0.5 mg per reaction. In the presence of chaperones, the recombinant L13 accumulates in soluble form. Expression of L13 was confirmed by Western blot and mass spectrometry. Surface plasmon resonance was used to characterize the function of the recombinant L13. Phlorizin was coupled to the sensor surface and used to investigate the binding ability. Preliminary results suggest that L13 indeed interact with phlorizin. Similarly high level expression of the extramembrane loop5 and loop7 separately or linked with each other and with loop13 was also achieved. Our data suggest that sufficient amount of soluble extramembrane loops of SGLT1 for structure-function relation analysis can be obtained by using the RTS 500 system.