Periplasmic expression of human interferon-α 2c in Escherichia coli results in a correctly folded molecule

• Published in: Biochemical journal 1994-03, Vol.298 (3), p.719-725
• Main Authors: Voss, T, Falkner, E, Ahorn, H, Krystek, E, Maurer-Fogy, I, Bodo, G, Hauptmann, R
• Format: Article
• Language: English
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2980719

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.