FIG. 7.

Sucrose density flotation gradient fractionation of three epitope-tagged BfpE derivatives. (A) Representations of epitope-tagged BfpE constructs. Each construct carries a C-terminal myc-His double epitope tag (not shown). For each construct, the number in the left column indicates the range of BfpE amino acids that are present. The middle column shows a linear representation of each protein. The right column displays the expected topology of each protein. Black boxes and cylinders indicate TM segments, while white boxes and cylinders indicate an exogenous N-terminal signal sequence, whose presumed removal is indicated by an arrow. (B) Lysates of TOP10 E. coli carrying plasmids producing the constructs shown in panel A were fractionated on sucrose density flotation gradients by centrifugation. Fractions were collected from the top (least dense portion) of the gradient, separated by SDS-PAGE on a 15% polyacrylamide gel, and subjected to immunoblotting using an antiserum recognizing the epitope tag. Fractions progress from the least dense on the left to the most dense on the right.