1. Periplasmic expression of human interferon-α 2c in Escherichia coli results in a correctly folded molecule
- Author
-
Horst Ahorn, Tilman Voss, R Hauptmann, G Bodo, E Krystek, I Maurer-Fogy, and E Falkner
- Subjects
Protein Folding ,Sequence analysis ,Blotting, Western ,Molecular Sequence Data ,Gene Expression ,Alpha interferon ,Enterotoxin ,Protein Sorting Signals ,Biology ,medicine.disease_cause ,Peptide Mapping ,Biochemistry ,Escherichia coli ,medicine ,Humans ,Amino Acid Sequence ,Promoter Regions, Genetic ,Molecular Biology ,Chromatography, High Pressure Liquid ,Chromatography ,Base Sequence ,Circular Dichroism ,Cell Biology ,Periplasmic space ,Alkaline Phosphatase ,Recombinant Proteins ,Fermentation ,Interferon Type I ,Alkaline phosphatase ,Research Article ,Cysteine - Abstract
Human interferon-alpha 2c (IFN-alpha 2c) was produced in Escherichia coli under the control of the alkaline phosphatase promoter using a periplasmic expression system. Compared with other leader sequences, the heat-stable enterotoxin II leader of E. coli (STII) resulted in the highest rate of correct processing as judged by Western-blot analysis. The fermentation was designed as a batch-fed process in order to obtain a high yield of biomass. The processing rate of IFN-alpha 2c could be increased from 25% to more than 50% by shifting the fermentation pH from 7.0 to 6.7. IFN-alpha 2c extracted from the periplasm was purified by a new four-step chromatographic procedure. Whereas cytoplasmically produced IFN-alpha 2c does not have its full native structure, IFN-alpha 2c extracted from the periplasm was found to be correctly folded, as shown by c.d. spectroscopy. Peptide-map analysis in combination with m.s. revealed the correct formation of disulphide bridges. N-terminal sequence analysis showed complete removal of the leader sequence, creating the authentic N-terminus starting with cysteine.
- Published
- 1994