Isolation and structural characterization of three isoforms of recombinant consensus alpha interferon.

Recombinant DNA-derived consensus alpha interferon was expressed in Escherichia coli and purified. Isoelectric focusing of this purified protein indicated the presence of three isoelectric subforms of pI 6.1, 6.0, and 5.7. These three subforms were preparatively separated by isoelectric focusing using Immobiline polyacrylamide gel and did not exhibit apparent differences in biological activity and tertiary structure. The pI 5.7 subform could also be separated from the pI 6.1 and 6.0 subforms by reverse-phase HPLC. Automated N-terminal amino acid sequence analysis of the pI 6.1 and 6.0 subforms yielded sequences corresponding to the methionyl and des-methionyl forms of the protein, respectively. Sequence analysis of the pI 5.7 subform indicated that its N terminus is blocked. To further determine the structure of the blocking moiety in the pI 5.7 subform, a blocked N-terminal tryptic peptide was isolated from HPLC peptide mapping of the S-carboxymethylated derivative. Results obtained from mass spectroscopic and amino acid analyses of this peptide suggest that it is blocked with an acetyl group at the N-terminal cysteine residue.

Pertussis holotoxoid formed in vitro with a genetically deactivated S1 subunit.

The cytotoxicity of pertussis toxin, a multisubunit exotoxin produced by Bordetella pertussis, is believed to be due to the ADP-ribosyltransferase activity of the S1 subunit. We have previously described the recombinant expression of each of the five individual pertussis toxin subunits in Escherichia coli and the production of an enzymatically deficient form of the S1 subunit by site-directed mutagenesis. We now report the in vitro assembly of holotoxin from native pertussis toxin B oligomer and recombinant S1 subunits, the latter purified and refolded from insoluble inclusion bodies. Holotoxin assembled with recombinant S1 of authentic amino acid sequence was indistinguishable from native pertussis toxin in its electrophoretic migration and ability to elicit a cytopathic response in cultured Chinese hamster ovary cells; in contrast, holotoxin assembled with the genetically deactivated analog of recombinant S1 displayed greatly diminished cytopathicity. These results verify that the in vitro cytopathic effects of pertussis toxin are the result of the enzymatic activity of the S1 subunit and illustrate the potential for constructing complex quaternary protein structures in vitro from insoluble, unfolded polypeptides derived from expression in recombinant systems.

Contribution of the B oligomer to the protective activity of genetically attenuated pertussis toxin.

An enzymatically deficient recombinant S1 subunit, in which Arg-9 was replaced by Lys, was combined with native B oligomer to form a mutant holotoxin molecule. This molecule exhibited decreased leukocytosis-promoting and histamine-sensitizing activities compared with those of the native toxin, supporting the view that the B oligomer is not responsible for these activities. The protective activity of this genetically attenuated pertussis toxin was compared with that of B oligomer alone. The mutant pertussis toxin and B oligomer were similarly capable of protecting mice against a respiratory infection with Bordetella pertussis, suggesting that the B oligomer makes a significant contribution to the protection afforded by the genetically attenuated holotoxin.