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. Study holds ProTherm entries: 5090, 5091 Extra Details: Met (1-190) refolding pathway; disulfide bonds; loop domain;,framework intermediates; recombinant proteins
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:28 p.m.
|Number of data points||5|
|Proteins||Growth hormone receptor|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: dG_H2O ; Derived Quantity: ddG_H2O|
|Libraries||Mutations for sequence MDLWQLLLTLAVAGSSDAFSGSEATPAVLVRASQSLQRVHPGLETNSSGKPKFTKCRSPELETFSCHWTDGVRHGLQSPGSIQLFYIRRSTQEWTQEWKECPDYVSAGENSCYFNSSYTSIWIPYCIKLTSNGGTVDQKCFSVEEIVQPDPPIGLNWTLLNISLTGIHADIQVRWEPPPNADVQKGWIVLEYELQYKEVNETQWKMMDPVLSTSVPVYSLRLDKEYEVRVRSRQRNSEKYGEFSEVLYVTLPQMSPFACEEDFRFPWFLIIIFGIFGLTVILFLLIFSKQQRIKMLILPPVPVPKIKGIDPDLLKEGKLEEVNTILAIHDNYKHEFYSDDSWVEFIELDIDDPDEKTEGSDTDRLLNNDHEKSLTILGAKEDDSGRTSCYEPDILETDFNANDVCDGTAEVAQPQRLKGEADLLCLDQKNQNNSPSNDAAPATQQPSVILAEENKPRPLIISGTDSTHQTAHTQLSNPSSLANIDFYAQVSDITPAGSVVLSPGQKNKAGISQCDMHLEVVSPCPANFIMDNAYFCEADAKKCIAMAPHVEVESRLAPSFNQEDIYITTESLTTTAGRSATAECAPSSEMPVPDYTSIHIVQSPQGLVLNATALPLPDKEFLSSCGYVSTDQLNKIMP|