Improved periplasmic production of biologically active murine interleukin-2 in Escherichia coli through a single amino acid change at the cleavage site

We fused the mature murine Interleukin-2 (mIL-2) gene to the signal peptide of the Outer membrane protein A (OmpA). We generated mutants mimicking different cleavage sites. A hybrid protein consisting of the OmpA signal peptide fused precisely to mature mIL-2, thereby mimicking the cleavage site of...

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Bibliographic Details
Published in:Process biochemistry (1991) 2006-06, Vol.41 (6), p.1343-1346
Main Authors: Robbens, Johan, De Coen, Wim, Fiers, Walter, Remaut, Erik
Format: Article
Language:English
Subjects:
E. coli
High-level production
Interleukin-2
Periplasm
Recombinant
Signal peptide
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Summary:We fused the mature murine Interleukin-2 (mIL-2) gene to the signal peptide of the Outer membrane protein A (OmpA). We generated mutants mimicking different cleavage sites. A hybrid protein consisting of the OmpA signal peptide fused precisely to mature mIL-2, thereby mimicking the cleavage site of the OmpA native protein, was very poorly secreted into the periplasm of Escherichia coli (200 U/ml). Insertion of a serine residue between the OmpA signal peptide and the mIL-2 mature sequence, thus mimicking the mIL-2 natural cleavage site, increased the secretion by a factor of 40,000 (8 × l0 6 U/ml). The specific biological activity of secreted mIL-2 equaled that of natural mIL-2 and was about five times higher than that of mIL-2 refolded from inclusion bodies. We also show that the temperature at which the culture is grown has a major impact on the secretion level.
ISSN:1359-5113
1873-3298
DOI:10.1016/j.procbio.2006.01.009