Bayle, D., Weeks, D. and Sachs, G.
Notes: The TNT® Coupled Reticulocyte Lysate System was used to characterize the membrane domain of several polytopic proteins. The two systems employed here use the presence or inhibition of glycosylation as an indicator of membrane insertion. The addition of a core glycosylation site resulted in an increase of ~2.5kDa to the relative molecular mass determined by SDS-PAGE. Using fusion vectors HK-M0 and HK-M1, the ability of each transmembrane domain to insert into the membrane was assessed. Of seven putative membrane segments, some had signal anchor properties, one had stop transfer properties and another had no membrane insertion activity. Combining data from the different systems confirmed seven transmembrane segments. (1649)