title: Improved recovery of active GST-fusion proteins from insoluble aggregates: solubilization and purification conditions using PKM2 and HtrA2 as model proteins.
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.
authors.affiliation: Department of Biomedical Sciences, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea.
authors.forename: Dae-Wook
authors.initials: DW
authors.lastname: Park
authors.forename: Sang-Soo
authors.initials: SS
authors.lastname: Kim
authors.forename: Min-Kyung
authors.initials: MK
authors.lastname: Nam
authors.forename: Goo-Young
authors.initials: GY
authors.lastname: Kim
authors.forename: Jungho
authors.initials: J
authors.lastname: Kim
authors.forename: Hyangshuk
authors.initials: H
authors.lastname: Rhim
mesh_terms: D000818:Animals; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000074923:High-Temperature Requirement A Serine Peptidase 2; D051379:Mice; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D017830:Octoxynol / Q000737:chemistry; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012995:Solubility
chemical_list: D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D017830:Octoxynol; D005982:Glutathione Transferase; D011770:Pyruvate Kinase; D012697:Serine Endopeptidases; D000074923:High-Temperature Requirement A Serine Peptidase 2; C000615915:Htra2 protein, mouse