Synthetic multifunctional proteins: isolation of covalently linked tryptophan synthetase alpha-subunit-lac-repressor-beta-galactosidase chimeras

Mol Gen Genet. 1977 Oct 24;155(3):301-7. doi: 10.1007/BF00272809.

Abstract

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.

MeSH terms

  • Bacterial Proteins*
  • Enzyme Precursors
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Escherichia coli / isolation & purification
  • Genes, Regulator
  • Lactose / metabolism
  • Molecular Weight
  • Mutation
  • Operon
  • Phenotype
  • Protein Multimerization
  • Tryptophan Synthase
  • beta-Galactosidase

Substances

  • Bacterial Proteins
  • Enzyme Precursors
  • beta-Galactosidase
  • Tryptophan Synthase
  • Lactose