Effect of environment on insulin-like growth factor I refolding selectivity.
Recombinant human insulin-like growth factor I (IGF-I), a 70-amino-acid peptide containing three disulphide bonds, produces two monomeric and several multimeric species during refolding. To optimize production of correctly folded IGF-I, conditions which influence protein refolding, stability and solubility were systematically examined. Combinations of solution components and conditions were analysed to identify synergistic interactions which enhance or reduce refolding efficiency. IGF-I concentration had the largest effect on formation of correctly folded peptide, due to competing association reactions. Solution polarity had the next largest individual effect, both on the level of multimeric peptide and on the relative proportion of correctly folded to misfolded monomeric peptide. Salt type and concentration and chaotrope type and concentration also had large individual effects on the distribution of IGF-I forms produced. Solution polarity modulated the effects of many other conditions including chaotrope concentration, salt type and concentration, and osmolyte concentration. Simultaneously decreasing the solution polarity and increasing the salt concentration improved the yield of correctly folded IGF-I relative to either individual change. Optimum solution conditions for refolding were 2M urea, 1M NaCl and 20% (v/v) ethanol. A possible mechanism for the importance of solution polarity on IGF-I refolding is discussed.
Hart
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Department of Recovery Process R&D, Genentech Inc., South San Francisco, CA 94080-4918.
Giltinan
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Lester
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PM
Reifsnyder
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H
Ogez
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JR
Builder
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D000595:Amino Acid Sequence; D019791:Guanidine; D006146:Guanidines; D007334:Insulin-Like Growth Factor I / Q000737:chemistry*; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D012492:Salts / Q000032:analysis; D012996:Solutions; D013696:Temperature; D014508:Urea
D006146:Guanidines; D011994:Recombinant Proteins; D012492:Salts; D012996:Solutions; D007334:Insulin-Like Growth Factor I; D014508:Urea; D019791:Guanidine