Solubilization of Recombinant Ovine Growth Hormone with Retention of Native-like Secondary Structure and Its Refolding from the Inclusion Bodies of Escherichia coli

• Published in: Biotechnology progress Vol. 14; no. 5; pp. 722 - 728
• Main Authors: Khan, R. H., Rao, K. B. C. Appa, Eshwari, A. N. S., Totey, S. M., Panda, A. K.
• Format: Journal Article
• Language: English
• Published: USA American Chemical Society 01.09.1998; American Institute of Chemical Engineers
• Subjects: Animals; Batch cell culture; Biological and medical sciences; Biotechnology; Chromatography, Gel; Column chromatography; Conformations; Escherichia coli; Escherichia coli - genetics; Fermentation; Fundamental and applied biological sciences. Psychology; Gene Expression; Growth Hormone - chemistry; Growth Hormone - genetics; Growth Hormone - isolation & purification; Health. Pharmaceutical industry; Hormones; Hormones. Growth factors; Hydrogen-Ion Concentration; Immunology; Inclusion Bodies - chemistry; Industrial applications and implications. Economical aspects; Molecular structure; Production of active biomolecules; Protein Folding; Protein Structure, Secondary; Purification; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Sheep; Solubility; Recombinant microorganism; Purification; Escherichia coli; Refolding; Somatotropin hormone; Gene expression; Fermentation; Solubilization; Secondary structure; Protein hormone; Vertebrata; Mammalia; Production; Bacteria; Sheep; Artiodactyla; Inclusion body; Recombinant protein; Vector; Ungulata; Enterobacteriaceae
• ISSN: 8756-7938; 1520-6033
• DOI: 10.1021/bp980071q

Ovine growth hormone was expressed in Escherichia coli in the form of inclusion bodies using the pQE‐30 expression vector. In a simple fed‐batch fermentation, 800 mg/L of recombinant ovine growth hormone (r‐oGH) was produced at a cell concentration of 12 g dry cell weight/L. Inclusion bodies were isolated from cells with >95% purity by extensive washing using detergent, and the r‐oGH from the purified inclusion bodies was solubilized in 2 M Tris‐HCl buffer at pH 12 containing 2 M urea. The r‐oGH solubilized in the above conditions exhibited considerable secondary structure as determined by circular dichroism spectra and was immunologically active. Solubilization of the inclusion body protein with retention of native‐like secondary structure gave higher yields during refolding. To suppress protein aggregation, refolding was carried out in gel filtration column. Refolding, buffer exchange, and the purification of monomeric r‐oGH from aggregated complex was achieved in a single step using gel filtration chromatography. More than 60% of the initial inclusion body protein was refolded into a native‐like conformation by the use of this procedure. The refolded protein was characterized by circular dichroism, fluorescence, SDS‐PAGE, Western blotting, and radio receptor binding assay and found to be similar to native, pituitary‐derived, ovine growth hormone.