Control of aggregation in protein refolding: Cooperative effects of artificial chaperone and cold temperature

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• 关键词：Aggregation ; Artificial chaperone ; Cyclodextrin ; Recombinant-human growth hormone
• 刊名：International Journal of Biological Macromolecules
• 出版年：2007
• 出版时间：30 January 2007
• 年：2007
• 卷：40
• 期：2
• 页码：126-133
• 全文大小：864 K

文摘

Refolding of GuHCl-denatured recombinant-human growth hormone (r-hGH) was investigated in both dilution additive and artificial chaperone assisted modes. In both techniques, it was found that CTAB is a better additive (in dilution mode) or a capturing agent (in artificial chaperone method). Neither of the two techniques was capable of complete inhibition of aggregates formed during refolding process. In dilution, using CTAB or α-cyclodextrin (α-CD) as two different additives, the aggregation was inhibited by almost 55 % . However, the extent of inhibition raised to almost 82 % in artificial chaperone assisted mode using CTAB as the capturing and α-CD as the stripping agents. Maximum inhibition of aggregation (up to 97 % ) was obtained when the entire process of refolding was done at 4 °C. However, under this temperature program, the far-UV CD and intrinsic fluorescence spectra of the refolded samples were not superimposable on their respective native spectra. The spectra superimposibilities were obtained when the refolding process was achieved under a well worked out temperature program: incubation of the sample for 3 min at 4 °C after initiation of the stripping step followed by overnight incubation at 22 °C. Based on these data, it is expected that higher activity recovery yields of recombinant proteins, particularly at relatively higher protein concentrations, could be achieved by getting a better molecular understanding of major factors responsive for aggregation and refolding pathways.