Lactose carrier protein, purification

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. 

Outline of purification procedure for lactose carrier protein from E. coli. 

The purification of two proteins, UMP synthase from mammalian tumor cells, and lactose carrier protein from E. coli bacteria is described in detail to illustrate how different fractionation methods can be combined most effectively. 

Methods for Purification and Characterization of Proteins, the primary goal is to acquaint the reader with the techniques used for protein purification. The first part of chapter 6 presents methods for protein fractionation. In the second part of this chapter, purification procedures for two proteins, UMP synthase and lactose carrier protein, are presented so that the student can see how different purification steps are combined for maximum effectiveness. 

In cases where it has proved impractical to assay for activity at each step, protein purification has been aided by tagging a fraction of the molecules with a photoaffinity reagent (e.g. the lactose carrier of Escherichia coli Newman et al., 1981 and the /J-adrenergic receptor of frog erythrocytes Shorr et al., 1982). 

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