Expression of ribonucleolytic toxin restrictocin in Escherichia coli: purification and characterization

Abstract

Restrictocin is a toxin produced by the fungusAspergillus restrictus. 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 restrictucin 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.

A Bispecific Enediyne-Energized Fusion Protein Containing Ligand-Based and Antibody-Based Oligopeptides against Epidermal Growth Factor Receptor and Human Epidermal Growth Factor Receptor 2 Shows Potent Antitumor Activity

“…1A, the full gene of fusion protein Ec-LDP-Hr (from 5′ to 3′) consisted of pelB signal peptide (22 amino acids; ref. 21), C-loop of EGF (the 22 amino acids of EGF COOH terminal), apoprotein LDP (110 amino acids; ref. 22) and V H CDR3 region of C6.5 antibody (20 amino acids; refs.…”

Binding capability of the enediyne-associated apoprotein to human tumors and constitution of a ligand oligopeptide-integrated protein

“…The synthesized gene of gldp was digested by Nde I/Xho I, and then ligated into pET-30a (+) digested with the same enzymes to give plasmid pET30GLDP. Then the G-LDP gene was amplified by the PCR from the pET30GLDP using direct primer (5 -GAATCCA CAT ATG AAA TAC CTG CTG CCG ACC GCT GCT GCT GGT CTG CTG CTC CTC GCT GCC CAG CCG GCG ATG GCC ATG GCC AAC CCC GTG GTG GGC TAC-3 ) including the signal peptide sequence (Rathore et al, 1996) and Nde I restriction site, as well as reverse primer (5 -GTTA CTC GAG GCC GAA GGT CAG AGC CAC GTG-3 ) including Xho I site. The synthesized gene of sgldp was digested by Nde I/Xho I, and then the released fragment gldp was ligated to pET-30a (+), generating recombinant plasmid pET30SGLDP.…”

Molecular Mechanism of Host Specificity in Plasmodium falciparum Infection

“…Supernatant, containing denatured protein, was collected, and the protein concentration was adjusted to 10 mg/ml with 6 M guanidine hydrochloride. Denatured protein was reduced by adding dithioerythritol, to a final concentration of 65 mM, and incubating at room temperature for 2 h. Protein was renatured by diluting 100-fold, in refolding buffer (100 mM Tris, pH 8.0, 0.5 M L-arginine-HCl, 2 mM EDTA, and 0.9 mM oxidized glutathione) (18). After incubating at 10°C for 36 h, renatured material was dialyzed against 20 mM Tris, pH 8.0, containing 100 mM urea.…”

“…Inclusion bodies were isolated from the spheroplast, solubilized using guanidine hydrochloride and reduced by adding dithioerythritol. The protein was subsequently renatured in vitro in a redox system using oxidized glutathione (18). The protein was purified to homogeneity by successive chromatography on anion exchange and gel filtration columns.…”