title: Obtaining Soluble Folded Proteins from Inclusion Bodies Using Sarkosyl, Triton X-100, and CHAPS: Application to LB and M9 Minimal Media.
abstract: This unit describes a straightforward and efficient method of using sarkosyl to solubilize and recover difficult recombinant proteins, such as GST- and His6 -tagged fusion proteins, that are overexpressed in E. coli. This protocol is especially useful for rescuing recombinant proteins overexpressed in M9 minimal medium. Sarkosyl added to lysis buffers helps with both protein solubility and cell lysis. Higher percentage sarkosyl (up to 10%) can extract >95% of soluble protein from inclusion bodies. In the case of sarkosyl-solubilized GST-fusion proteins, batch-mode affinity purification requires addition of a specific ratio of Triton X-100 and CHAPS, while sarkosyl-solubilized His6 -tagged fusion proteins can be directly purified on Ni(2+) resin columns. Proteins purified by this method could be widely used in biological assays, structure analysis and mass spectrum assay.
authors.affiliation: Department of Chemistry, George Washington University, Washington, D.C.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Massiah
authors.affiliation: Department of Chemistry, George Washington University, Washington, D.C.
authors.forename: Katharine M
authors.initials: KM
authors.lastname: Wright
authors.affiliation: Department of Chemistry, George Washington University, Washington, D.C.
authors.forename: Haijuan
authors.initials: H
authors.lastname: Du
mesh_terms: D001622:Betaine / Q000737:chemistry; D002793:Cholic Acids / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D006639:Histidine / Q000235:genetics; D002479:Inclusion Bodies / Q000737:chemistry*; D017830:Octoxynol / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012521:Sarcosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility; D013012:Sorbitol / Q000737:chemistry
chemical_list: D002793:Cholic Acids; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D001622:Betaine; D006639:Histidine; D013012:Sorbitol; C025231:sarkosyl; D017830:Octoxynol; D005982:Glutathione Transferase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D012521:Sarcosine
keywords: CHAPS; Glutathione S-transferase (GST); His6-tagged; Inclusion bodies; M9 minimal medium; Triton X-100; protein expression and purification; sarkosyl