Recombinant Human Insulin-Like Growth Factor II Expressed in Escherichia coli

• Published in: Bio/Technology 1987-10, Vol.5 (10), p.1047-1051
• Main Authors: Furman, Thomas C, Epp, Janet, Hsiung, Hansen M, Hoskins, JoAnn, Long, George L, Mendelsohn, Laurane G, Schoner, Brigitte, Smith, Dennis P, Smith, Michele C
• Format: Article
• Language: English
• Subjects: Agriculture; Bioinformatics; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; Fundamental and applied biological sciences. Psychology; Health. Pharmaceutical industry; Industrial applications and implications. Economical aspects; Life Sciences; Other active biomolecules; Production of active biomolecules; research-paper
• ISSN: 0733-222X; 1087-0156; 2331-3684; 1546-1696
• DOI: 10.1038/nbt1087-1047

Human Insulin-like growth factor II (IGF-II) was produced in Escherichia coli as a 113 amino acid chimeric protein, LE1-Met-IGF-II consisting of 45 N-terminal amino acids from the E. coli trp leader peptide and the trp E polypeptide, and 67 C-terminal amino acids for IGF-II. High-level expression of the fusion protein, LE1-Met-IGF-II, was obtained with pCZ21, a plasmid containing a thermoinducible runaway replicon and the kanamycin resistance marker from Tn 5 . In E. coli , LE1-Met-IGF-II formed granules that were isolated and cleaved with cyanogen bromide to liberate IGF-II. IGF-II was sulfitolyzed, purified by anion exchange chromatography, refolded through a disulfide interchange reaction, and further purified by reverse-phase HPLC and gel filtration. The biological activity of this recombinant human IGF-II was measured in a competitive protein binding assay for IGF-II and by its ability to stimulate amino isobutyric acid uptake and protein synthesis in NCTC 2414 fibroblast cultures.