Periplasmic expression of human interferon-alpha 2c in Escherichia coli results in a correctly folded molecule.
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.
Voss
T
T
Ernst-Boehringer Institut für Arzneimittelforschung, Bender & Co., Vienna, Austria.
Falkner
E
E
Ahorn
H
H
Krystek
E
E
Maurer-Fogy
I
I
Bodo
G
G
Hauptmann
R
R
D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics*; D005285:Fermentation; D015870:Gene Expression; D006801:Humans; D007370:Interferon Type I / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011401:Promoter Regions, Genetic; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000737:chemistry; D011994:Recombinant Proteins
D007370:Interferon Type I; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase