Overproduction of fungal ribotoxin alpha-sarcin in Escherichia coli: generation of an active immunotoxin.
alpha-Sarcin is a ribonucleolytic protein secreted by the mold Aspergillus giganteus. DNA encoding alpha-sarcin was isolated from the host and cloned into T7 promoter based E. coli expression vectors. Using bacterial outer membrane protein A (OmpA) signal sequence, properly processed recombinant (re-) protein was secreted into the culture medium while in the absence of a signal sequence protein remained insoluble in the bacterial inclusion bodies. The re-alpha-sarcin was purified to homogeneity by simple chromatographic techniques both from the insoluble and soluble sources with respective yields of 40-50 microg/ml and 2-3 microg/ml. The re-ribotoxin was functionally as active as the native toxin and preserved its specificity. The re-alpha-sarcin was used in the construction of an active immunotoxin targeted at the human cancer cells overexpressing transferrin receptor (TFR).
Rathore
D
D
Immunochemistry Laboratory, National Institute of Immunology, New Delhi, India.
Nayak
S K
SK
Batra
J K
JK
D001230:Aspergillus / Q000235:genetics*; D003001:Cloning, Molecular; D004722:Endoribonucleases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins; D000922:Immunotoxins / Q000235:genetics*
D005656:Fungal Proteins; D000922:Immunotoxins; C014801:alpha-sarcin; D004722:Endoribonucleases