Timothy D. Bartley

Structural characterization of recombinant consensus interferon-α

Abstract Recombinant consensus interferon-α is derived from genetically modified Escherichia coli containing a synthetic gene constructed from a consensus of interferon sequences. The purified and biologically active protein has been subjected to detailed structural characterization including sequence determination and peptide isolation and identification. The homogeneous consensus interferon-α preparation contains two chromatographically indistinguishable homologous polypeptides with one containing an extra methionyl residue at the amino terminus. The delineated amino acid sequence of the protein is identical to that expected from the coding sequence of the gene. Correct oxidation of the molecule has been confirmed with two intramolecular disulfide linkages observed at Cys(1)-Cys(99) and Cys(29)-Cys(139).

Isolation and structural characterization of three isoforms of recombinant consensus α 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.

Carbohydrate structure characterization of two soluble forms of a ligand for the ECK receptor tyrosine kinase

Publisher Summary Characterization of glycoproteins requires application of several chromatographic and mass spectrometric analytical techniques. This chapter discusses carbohydrate microheterogeneity using high pH anion exchange chromatography, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and electrospray ionization mass spectrometry (ES-MS) after isolation and identification of the peptides containing glycosylation sites. MALDI-TOF mass spectrometry generally gives good results for the glycopeptides at sub-picomolar to low picomolar levels, and it is possible to analyze nearly all of the peptides using this technique. The purified glycopeptides are analyzed by ES-MS and in several cases produced some different ion populations than what is observed in the MALDI-TOF spectra. Based on the mass spectrometric and high pH anion exchange chromatographic results, the major difference between the two r-HuB61 150 forms secreted from CHO cells is in the type of N-linked carbohydrate glycosylation at Asn 8 . The partial glycosylation pattern observed in this study is also common to other recombinant glycoproteins produced in CHO cells, such as stem cell factor.

Developments toward a Recombinant Pertussis Vaccine

Vaccines produced from chemically “inactivated” Bordetella pertussis have been remarkably effective in diminishing the incidence of whooping cough in the developed world (Hinman and Koplan, 1984). The frequency of adverse vaccine reactions attributed to pertussis immunization, however, has led to a reassessment of the vaccine material and its components (Hinman and Onorato, 1987). In an effort to provide a less reactogenic vaccine, we have focused on a major virulence factor of the Gram-negative etiologic agent. Pertussis toxin (PTX ), also known as lymphocytosis promotion factor (LPF), islet activating protein (IAP), histamine sensitization factor (HSF), and pertussigen (Munoz, 1985), has been shown to be both an important mediator of pertussis disease (Pittman, 1979) and an effective protective element in acellular vaccines that have reduced reactogenicity in pediatric vaccine recipients (Manclark and Cowell, 1984; Robinson et al., 1985; Kallings et al.,1988).