Isolation and Characterization of Mutants of Escherichia coli K12 Affected in Protein Localization

This chapter discusses the isolation and characterization of mutants of Escherichia coli K12 affected in protein localization. The chapter discusses the techniques that are used for isolation and characterization of gene fusions and describes various procedures that can be used to isolate mutants that exhibit an alteration in the export process. The most useful gene fusions for studying protein localization are those that produce a hybrid protein comprising an NH₂-terminal fragment of an exported protein and a carboxylic acid (COOH)-terminal fragment of a protein to serve as a label for biochemical identification of the hybrid molecule. With the advent of recombinant DNA (rDNA) techniques, any one of a number of different proteins could be chosen to serve as a label. The chapter illustrates the use of enzyme β-galactosidase. The nature of the enzyme β-galactosidase distinguishes lactose metabolism from most other biochemical pathways and makes it easy to analyze. β-galactosidase is particularly amenable to gene fusion techniques because the NH₂-terminal portion of the enzyme can be removed and replaced by an NH₂-terminal fragment from any other protein, and yet the hybrid can retain β-galactosidase activity.