An efficient expression system for a variant form of the cytotoxic protein alpha-sarcin in Escherichia coli

Biochim Biophys Acta. 1992 Mar 24;1130(2):182-8. doi: 10.1016/0167-4781(92)90526-6.

Abstract

An efficient Escherichia coli system for the production of a variant form of the cytotoxic protein alpha-sarcin (delta Ala 1) has been constructed. cDNA encoded alpha-sarcin lacking N-terminal alanine was ligated with the bla signal peptide sequence (the signal sequence of E. coli beta-lactamase localizes the protein in the periplasm) and was inserted into an inducible bacterial expression vector pKTN2-2. When the plasmid introduced into E. coli was expressed in the presence of IPTG, the recombinant product accumulated in the periplasmic space. The product was purified by Affi-Gel Blue followed by Bio-Rex 70 column chromatography. Recombinant alpha-sarcin (delta Ala 1) displayed the properties similar to those of authentic alpha-sarcin isolated from Aspergillus giganteus with respect to its molecular mass and enzymatic activity in ribosomal inactivation. The amount of alpha-sarcin variant produced in the system was estimated to be 1.2 mg/l of culture.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Blotting, Western
  • Cloning, Molecular / methods*
  • DNA / genetics
  • Electrophoresis, Polyacrylamide Gel
  • Endoribonucleases*
  • Escherichia coli / genetics*
  • Fungal Proteins / genetics*
  • Fungal Proteins / isolation & purification
  • Fungal Proteins / metabolism
  • Gene Expression
  • Genetic Vectors
  • Molecular Sequence Data
  • Plasmids
  • Protein Synthesis Inhibitors / isolation & purification
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification

Substances

  • Fungal Proteins
  • Protein Synthesis Inhibitors
  • Recombinant Proteins
  • alpha-sarcin
  • DNA
  • Endoribonucleases