Expression of ribonucleolytic toxin restrictocin in Escherichia coli: purification and characterization.
Restrictocin is a toxin produced by the fungus Aspergillus restritus. The DNA coding for restrictocin was isolated from the host by polymerase chain reaction and cloned into a T7 promoter-based expression vector. The protein was overproduced in Escherichia coli and remained insoluble in the cell in the form of inclusion bodies. Recombinant restrictocin was purified in large amounts, by a simple denaturation-renaturation protocol involving a redox system, with typical yields of 45 mg/l of original culture. Restrictocin could be secreted into the bacterial medium using ompA, pelB and LTB signal sequences. Among the three signal sequences, ompA was found to be the most efficient in secreting the recombinant protein. The protein secreted into the extracellular medium was properly processed as evident by the amino-terminal sequencing. Recombinant restrictocin was readily purified to homogeneity from either the medium or inclusion bodies by simple chromatographic techniques and was found to be functionally as active as the native fungal protein in inhibiting the eukaryotic translation.
Rathore
D
D
Immunochemistry Laboratory, National Institute of Immunology, New Delhi, India.
Nayak
S K
SK
Batra
J K
JK
D000485:Allergens; D000595:Amino Acid Sequence; D000818:Animals; D052179:Antigens, Plant; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D005914:Globins / Q000037:antagonists & inhibitors; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012156:Reticulocytes / Q000187:drug effects; D012260:Ribonucleases / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology
C081947:ASPF1 protein, Aspergillus fumigatus; D000485:Allergens; D052179:Antigens, Plant; D005656:Fungal Proteins; D011994:Recombinant Proteins; C036196:MITF protein, Aspergillus restrictus; D005914:Globins; D012260:Ribonucleases