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http://dx.doi.org/10.5483/BMBRep.2011.44.4.279

Improved recovery of active GST-fusion proteins from insoluble aggregates: solubilization and purification conditions using PKM2 and HtrA2 as model proteins  

Park, Dae-Wook (Department of Biomedical Sciences, College of Medicine, The Catholic University of Korea)
Kim, Sang-Soo (Department of Biomedical Sciences, College of Medicine, The Catholic University of Korea)
Nam, Min-Kyung (Department of Biomedical Sciences, College of Medicine, The Catholic University of Korea)
Kim, Goo-Young (Department of Biomedical Sciences, College of Medicine, The Catholic University of Korea)
Kim, Jung-Ho (Department of Life Science, Sogang University)
Rhim, Hyang-Shuk (Department of Biomedical Sciences, College of Medicine, The Catholic University of Korea)
Publication Information
BMB Reports / v.44, no.4, 2011 , pp. 279-284 More about this Journal
Abstract
The glutathione S-transferase (GST) system is useful for increasing protein solubility and purifying soluble GST fusion proteins. However, purifying half of the GST fusion proteins is still difficult, because they are virtually insoluble under non-denaturing conditions. To optimize a simple and rapid purification condition for GST-pyruvate kinase muscle 2 (GST-PKM2) protein, we used 1% sarkosyl for lysis and a 1 : 200 ratio of sarkosyl to Triton X-100 (S-T) for purification. We purified the GST-PKM2 protein with a high yield, approximately 5 mg/L culture, which was 33 times higher than that prepared using a conventional method. Notably, the GST-high-temperature requirement A2 (GST-HtrA2) protein, used as a model protein for functional activity, fully maintained its proteolytic activity, even when purified under our S-T condition. This method may be useful to apply to other biologically important proteins that become highly insoluble in the prokaryotic expression system.
Keywords
Insoluble GST-fusion protein; Prokaryotic expression system; Sarkosyl; Solubilization; Triton X-100;
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