Solubilization of recombinant ovine growth hormone with retention of native-like secondary structure and its refolding from the inclusion bodies of Escherichia coli.
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.
Product Development Cell and Embryo Biotechnology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110 067, India.
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RH
Khan
K B
KB
Rao
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AN
Eshwari
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SM
Totey
A K
AK
Panda
D000818:Animals; D001709:Biotechnology; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D013006:Growth Hormone / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012756:Sheep; D012995:Solubility
D011994:Recombinant Proteins; D013006:Growth Hormone