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.
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.
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Centre for Animal Biotechnology, School of Veterinary Science, University of Melbourne, Parkville, Victoria, Australia.
Cardamone
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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
D002593:Cetrimonium Compounds; D006146:Guanidines; D011994:Recombinant Proteins; D014508:Urea; D013006:Growth Hormone; D019791:Guanidine; D000077286:Cetrimonium