A screening system for the identification of refolding conditions for a model protein kinase, p38α

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• 作者：Richard H. Cowan ; Rick A. Davies ; Teresa T.J. Pinheiro
• 关键词：Protein kinases ; Protein folding ; Folding additives ; Inclusion bodies ; Refolding ; Recombinant protein expression
• 刊名：Analytical Biochemistry
• 出版年：2008
• 出版时间：1 May 2008
• 年：2008
• 卷：376
• 期：1
• 页码：25-38
• 全文大小：644 K

文摘

Protein kinases are key drug targets involved in the regulation of a wide variety of cellular processes. To aid the development of drugs targeting these kinases, it is necessary to express recombinant protein in large amounts. The expression of these kinases in Escherichia coli often leads to the accumulation of the expressed protein as insoluble inclusion bodies. The refolding of these inclusion bodies could provide a route to soluble protein, but there is little reported success in this area. We set out to develop a system for the screening of refolding conditions for a model protein kinase, p38rc=""http://www.sciencedirect.com/scidirimg/entities/204e.gif"" alt=""greek small letter alpha"" title=""greek small letter alpha"" border=""0"">, and applied this system to denatured p38rc=""http://www.sciencedirect.com/scidirimg/entities/204e.gif"" alt=""greek small letter alpha"" title=""greek small letter alpha"" border=""0""> derived from natively folded and inclusion body protein. Clear differences were observed in the refolding yields obtained, suggesting differences in the folded state of these preparations. Using the screening system, we have established conditions under which soluble, folded p38rc=""http://www.sciencedirect.com/scidirimg/entities/204e.gif"" alt=""greek small letter alpha"" title=""greek small letter alpha"" border=""0""> can be produced from inclusion bodies. We have shown that the refolding yields obtained in this screen are suitable for the economic large-scale production of refolded p38rc=""http://www.sciencedirect.com/scidirimg/entities/204e.gif"" alt=""greek small letter alpha"" title=""greek small letter alpha"" border=""0""> protein kinase.