High-level direct expression of semi-synthetic human interleukin-6 in Escherichia coli and production of N-terminus met-free product.

We have developed a direct expression system for high-level production of recombinant human interleukin-6 (rhIL-6) in Escherichia coli. In this system, (i) the natural N-terminal coding region of the hIL-6 gene was replaced by a synthetic sequence containing A-T rich codons, (ii) dual Shine-Dalgarno (SD) sequences were employed, (iii) an A-T rich segment was inserted in front of the initiation codon to avoid putative mRNA secondary structure in the region and (iv) the natural amber termination codon of the hIL-6 gene was changed to an ocher stop codon. The hIL-6 polypeptide, synthesized at a high level, formed cytoplasmic inclusion bodies. After refolding, the N-terminal methionine was removed by aminopeptidase-P in vitro. The purified recombinant hIL-6 had B-cell differentiation activity equivalent to natural IL-6 from a human T-cell culture.