An efficient expression system for a variant form of the cytotoxic protein alpha-sarcin in Escherichia coli.
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.
Oka
T
T
Department of Nutritional Chemistry, School of Medicine, University of Tokushima, Japan.
Aoyama
Y
Y
Natori
Y
Y
Katano
T
T
Endo
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases; D004926:Escherichia coli / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D011500:Protein Synthesis Inhibitors / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D005656:Fungal Proteins; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; C014801:alpha-sarcin; D004247:DNA; D004722:Endoribonucleases