title: A relationship between the starting secondary structure of recombinant porcine growth hormone solubilised from inclusion bodies and the yield of native (monomeric) protein after in vitro refolding.
abstract: Recombinant porcine growth hormone (rPGH) was solubilised from inclusion bodies (IB's) using either 6 M guanidinium hydrochloride (GnHCl), 7.5 M urea or by a novel method using a cationic surfactant, cetyltrimethylammonium chloride (CTAC). Circular dichroism (CD) analysis of the secondary (2 degrees) structure of the urea- and GnHCl-solubilised rPGH showed the absence of alpha-helical content with the majority of the molecule existing in a 'random coil' structure. In contrast, the CTAC-solubilised rPGH displayed significant starting 2 degrees structure (10-15% alpha helix; 30-40% beta structure). The three rPGH preparations were refolded in vitro against weak urea. GnHCl or aqueous buffers, resulting in an average refolding efficiency of 50% native (monomeric) rPGH for CTAC solubilised IB's and only 20% for urea or GnHCl solubilised IB's. We conclude that the method of solubilisation of IB's and the resultant difference in the starting 2 degrees structure of rPGH, particularly alpha-helical content, is a major in vitro factor that apparently predetermines the aggregation/refolding behaviour rPGH irrespective of refolding environment.
authors.affiliation: Centre for Animal Biotechnology, School of Veterinary Science, University of Melbourne, Parkville, Victoria, Australia.
authors.forename: N K
authors.initials: NK
authors.lastname: Puri
authors.forename: M
authors.initials: M
authors.lastname: Cardamone
mesh_terms: D000818:Animals; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D013006:Growth Hormone / Q000737:chemistry* / Q000302:isolation & purification; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility; D013552:Swine; D014508:Urea / Q000737:chemistry
chemical_list: D002593:Cetrimonium Compounds; D006146:Guanidines; D011994:Recombinant Proteins; D014508:Urea; D013006:Growth Hormone; D019791:Guanidine; D000077286:Cetrimonium