A SIMPLE SCREEN FOR PERMISSIVE SITES IN PROTEINS - ANALYSIS OF ESCHERICHIA-COLI LAC PERMEASE

Proteins can be remarkably tolerant of major mutational changes. Sites that accomodate large insertions without loss of function (''permissi ve'' sites) appear generally to correspond to surface regions at which the added sequences do not disrupt overall folding. The identificatio n of such sites can aid in the engineering of functional derivatives o f a protein with novel properties. To screen for permissive sites, we developed a simple two-step procedure for generating 31-codon insertio ns in cloned genes. In a first step, a beta-galactosidase or alkaline phosphatase gene fusion is generated by insertion of a transposon deri vative into the target gene. Requiring beta-galactosidase or alkaline phosphatase activity fixes the translational reading frame of the tran sposon relative to the target gene. In a second step, most of the tran sposon sequences are excised in vitro, leaving the in-frame insertion. Insertions may be targeted either to cytoplasmic or exported protein sequences, and the inserted sequence acts as an epitope in a variety o f proteins. As a test case, a set of 31-codon insertions in the Escher ichia coli lac permease gene was generated. The lactose transport acti vities of the mutant proteins followed a simple pattern: most of the p roteins (10/12) with insertions in sequences thought to face the cytop lasm or periplasm were at least partially active, whereas all proteins (9/9) with insertions in membrane-spanning sequences were inactive. T he only exceptions were two inactive proteins with insertions in the t hird cytoplasmic region. Most of the inactive proteins were detected a t reduced levels in cells, presumably due to proteolytic breakdown. Th ese studies thus illustrate the use of the new method to identify perm issive sites and help document the remarkable sequence flexibility of many of the hydrophilic loops in lac permease. In addition to screenin g for permissive sites, 31-codon insertion mutagenesis may be useful i n epitope-tagging proteins at multiple internal positions, in analyzin g membrane protein topology, and in dissecting structure-function rela tionships in proteins. (C) 1997 Academic Press Limited.