Purification of a Membrane-Bound Protein

1 I The second purification procedure we examine illus- Vri trates an unusual approach to the purification of a membrane-bound protein. The lactose carrier protein of E. coli is normally tightly bound to the plasma membrane. This protein is involved in the active transport of the dissaccharide lactose across the cytoplasmic membrane. When lactose carrier protein is present, the intracellular concentration of lactose can achieve levels 1,000-fold higher than those found in the external medium. Ron Kaback devised a simple yet elegant procedure for the purification of this protein. 

Purification of the membrane-bound lactose carrier protein is a very different problem from the purification of the soluble OMP synthase. Both the approach to purification and the assays for the protein during purification are quite novel. The assay involves reconstituting a transport system with membranes that are free of lactose carrier protein, then adding the partially purified carrier protein and radioactively labeled lactose. The activity in this assay system is proportional to the transport of radioactive lactose across the membrane in the cell-free reconstituted system. 

The results of the purification steps are tabulated in table 6.5, and the purification procedure is outlined in 

Fraction Protein (tug) Percent Recovery llutal Protein) Ptrwnt Rccuv n (Carrier Protein) Purification Factor 

At this point, the carrier protein was released from a suspension of the membranes by addition of the hydrophobic reagent octylglucoside in the presence of E. coli phos- 

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