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.
Yasueda
H
H
Central Research Laboratories, Ajinomoto Co., Inc., Kawasaki, Japan.
Nagase
K
K
Hosoda
A
A
Akiyama
Y
Y
Yamada
K
K
D001402:B-Lymphocytes / Q000187:drug effects; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005813:Genes, Synthetic; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects; D008715:Methionine / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D014162:Transfection
D015850:Interleukin-6; D011994:Recombinant Proteins; D008715:Methionine