Comparing the Refolding and Reoxidation of Recombinant Porcine Growth Hormone from a Urea Denatured State and from Escherichia coli Inclusion Bodies

Abstract

Overexpression of cloned genes in bacteria often leads to insoluble refractile body formation requiring solubilization and refolding to obtain biologically active proteins. A refolding pathway was established for a model protein, porcine growth hormone (PGH), yielding an appreciably high recovery of 85%. The conditions include the dilution of a urea, beta-mercaptoethanol (beta-ME) denatured PGH solution in a refolding environment containing 3.5 M urea and 10 mM beta-ME/HED at a 10:1 ratio at pH 9.1 and 0.5 mg/mL PGH. The intrinsic fluorescence-detected transition of PGH in urea gives 3.8 kcal/mol for the free energy of denaturation (delta GH2O) of PGH. The native-like conformation of PGH is dependent on disulfide bonds because reduced and carboxymethylated PGH is devoid of tertiary structure as assessed by intrinsic tryptophan fluorescence. Physical analysis of C-terminally truncated recombinant PGH indicated no significant difference in the free energy of denaturation of P-band in urea as full-length PGH. This suggests that the first disulfide, forming the large loop domain of PGH, provides a significantly greater contribution to the conformational stability of PGH than the second disulfide, which forms the carboxy-terminal small loop domain. The rate of formation of native structure during refolding was biphasic, with native structure identified by intrinsic fluorescence and hydrophobicity spectroscopy prior to disulfide bond formation. Thus "framework" intermediates are prerequisites for correct disulfide formation and tertiary folding of PGH. This study shows how a protein refolds, forms disulfides, and self-associates, which may be useful for examining the refolding of other recombinant proteins.

Optimization of Inclusion Body Solubilization and Renaturation of Recombinant Human Growth Hormone from Escherichia coli

“…Chaotropic agents such as urea, guanidine hydrochloride (Gdn-HCl) 2 , and thiocyanate salts (8,9), detergents such as sodium dodecyl sulfate (SDS) (10), N-cetyltrimethylammonium chloride (11) and sarkosyl (sodium N-lauroyl sarcosine; NLS) (12) along with reducing agents like ␤-mercaptoethanol, dithiothreitol, or cysteine have been extensively used for solubilizing the inclusion body proteins. The soluble proteins are then refolded to their native state after the chaotropic agents or other salts are removed by dialyzing the proteins in buffers containing reducing and oxidizing agents (8,13).…”

Disulfide Bond Engineering to Monitor Conformational Opening of Apolipophorin III During Lipid Binding

“…We report here studies designed to test the validity of the hinge opening hypothesis by using a strategy that tethers the helix bundle by means of a disulfide bond and is hypothesized to prevent its proposed opening during lipoprotein interaction (Breiter et al, 1991). Site-directed introduction of non-natural intramolecular disulfide bonds has proven to be a valuable tool for obtaining information about protein conformational changes (Duche et al, 1994;Chang and Cronan, 1995), folding (Cardamone et al, 1995), stability (Wetzel et al, 1988;, and proximity relationships (Wolff-Long et al, 1995). We have introduced two cysteines into M. sexta apoLp-III (which otherwise lacks cysteine) by site-directed mutagenesis at strategic locations postulated to facilitate and maximize the probability of intramolecular disulfide bond formation.…”

Production of a recombinant human basic fibroblast growth factor with a collagen binding domain

“…The renaturation of protein aggregates in bacterial inclusion bodies into biologically active conformations involves a number of interdependent steps: solubilization, oxidative refolding, and withdrawal of denaturants, each of which represents a challenge of strategy and optimization (Handl et al 1993, Cardamone et al 1995. In the present study, we determined that a strong ionic denaturant, 6 M guanidine, was more effective than 8 M urea in solubilizing the inclusion bodies.…”

“…However, in evaluating the recoveries from subsequent refolding steps, we found that the rhbFGF activity recovered from guanidine-HC1 solutions was much lower than from urea. Others also have reported that guanidine-HC1 may interfere with the refolding process (Cardamone et al 1995). For this reason, we utilized guanidine-HC1 initially as a denaturant to solubilize the inclusion bodies and optimize the recovery of the recombinant proteins and then replaced the 6 M guanidine-HCt with 8 M urea in the course of affinity purification.…”