title: Purification of GST-Tagged Proteins.
abstract: This protocol describes the purification of recombinant proteins fused to glutathione S-transferase (GST, GST-tagged proteins) by Glutathione Affinity purification. The GST tag frequently increases the solubility of the fused protein of interest and thus enables its purification and subsequent functional characterization. The GST-tagged protein specifically binds to glutathione immobilized to a matrix (e.g., agarose) and can be easily separated from a cell lysate by a bind-wash-elute procedure. GST-tagged proteins are often used to study protein-protein interactions, again making use of glutathione affinity in a procedure called a GST pull-down assay. The protocol is designed to process 200 ml of E. coli culture expressing intermediate to high amounts of a GST-tagged protein (~25 mg l(-1)). Depending on the expression rate or the available culture volume, the scale can be increased or decreased linearly. The protocol can also be used to purify GST-tagged proteins from other expression systems, such as insect or mammalian cells. Tips are provided to aid in modifying certain steps if proteins shall be recovered from alternative expression systems.
authors.affiliation: QIAGEN GmbH, Research and Development, Qiagenstrasse 1, 40724 Hilden, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Schäfer
authors.affiliation: QIAGEN GmbH, Research and Development, Qiagenstrasse 1, 40724 Hilden, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Seip
authors.affiliation: QIAGEN GmbH, Research and Development, Qiagenstrasse 1, 40724 Hilden, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Maertens
authors.affiliation: QIAGEN GmbH, Research and Development, Qiagenstrasse 1, 40724 Hilden, Germany.
authors.forename: Helena
authors.initials: H
authors.lastname: Block
authors.affiliation: QIAGEN GmbH, Research and Development, Qiagenstrasse 1, 40724 Hilden, Germany. Electronic address: jan.kubicek@qiagen.com.
authors.forename: Jan
authors.initials: J
authors.lastname: Kubicek
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism*; D006801:Humans; D007313:Insecta; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012995:Solubility; D013696:Temperature
chemical_list: D011506:Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D005978:Glutathione
keywords: Bind-wash-elute; E. coli lysate; GST pull-down assay; GST-tagged proteins; Glutathione affinity chromatography; Glutathione affinity purification; PBS