Solubilization of recombinant ovine growth hormone with retention of native-like secondary structure and its refolding from the inclusion bodies of Escherichia coli
Rh. Khan et al., Solubilization of recombinant ovine growth hormone with retention of native-like secondary structure and its refolding from the inclusion bodies of Escherichia coli, BIOTECH PR, 14(5), 1998, pp. 722-728
Ovine growth hormone was expressed in Escherichia coli in the form of inclu
sion bodies using the pQE-30 expression vector. In a simple fed-batch ferme
ntation, 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 is
olated from cells with >95% purity by extensive washing using detergent, an
d 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 cir
cular dichroism spectra and was immunologically active. Solubilization of t
he inclusion body protein with retention of native-like secondary structure
gave higher yields during refolding. To suppress protein aggregation, refo
lding was carried out in gel filtration column. Refolding, buffer exchange,
and the purification of monomeric r-oGH from aggregated complex was achiev
ed in a single step using gel filtration chromatography. More than 60% of t
he initial inclusion body protein was refolded into a native-like conformat
ion by the use of this procedure. The refolded protein was characterized by
circular dichroism, fluorescence, SDS-PAGE, Western blotting, and radio re
ceptor binding assay and found to be similar to native, pituitary-derived,
ovine growth hormone.