Synthetic multifunctional proteins: isolation of covalently linked tryptophan synthetase alpha-subunit-lac-repressor-beta-galactosidase chimeras.
Several E. coli mutants were isolated which produce triple chimeras between one of the trp enzymes lac, repressor and beta-galactosidase. The mutants were isolated as TonB- Lac+ derivatives of a phenotypically Lac- TrpR- strain carrying a lac I+ -Z+ fusion on a phi80dlac phage. The phage is integrated into the chromosome in such a way that the lac and the trp genes are transcribed in the same direction. Of a total of 58 candidates 2 TrpA- and 3 Trp- strains produce triple chimeras. The chimeras from the two TrpA- strians were further examined. They consist of tryptophan synthetase alpha-subunit, lac repressor and beta-galactosidase. In crude extracts of these strains the tryptophan synthetase alpha-subunit part can be identified by its ability to aggregate with the beta-subunit since some of the beta-subunit activity can be precipitated with antiserum against beta-galactosidase. Furthermore beta-galactosidase precipitates with antiserum against tryptophan synthetase alpha-subunit. The lac repressor part is able to bind IPTG, but not lac operator DNA in vitro. The beta-galactosidase part is as unaffected as in the original lac repressor-beta-galactosidase chimera. The molecular weights of both chimeras are 175,000 when determined by SDS gel electrophoresis. The chimeras are partially degraded giving rise to fragments of distinct molecular weights.
D001426:Bacterial Proteins; D004792:Enzyme Precursors; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D005809:Genes, Regulator; D007785:Lactose / Q000378:metabolism; D008970:Molecular Weight; D009154:Mutation; D009876:Operon; D010641:Phenotype; D055503:Protein Multimerization; D014367:Tryptophan Synthase; D001616:beta-Galactosidase
D001426:Bacterial Proteins; D004792:Enzyme Precursors; D001616:beta-Galactosidase; D014367:Tryptophan Synthase; D007785:Lactose