title: A general path for large-scale solubilization of cellular proteins: from membrane receptors to multiprotein complexes.
abstract: Expression of recombinant proteins in bacterial or eukaryotic systems often results in aggregation rendering them unavailable for biochemical or structural studies. Protein aggregation is a costly problem for biomedical research. It forces research laboratories and the biomedical industry to search for alternative, more soluble, non-human proteins and limits the number of potential "druggable" targets. In this study we present a highly reproducible protocol that introduces the systematic use of an extensive number of detergents to solubilize aggregated proteins expressed in bacterial and eukaryotic systems. We validate the usefulness of this protocol by solubilizing traditionally difficult human protein targets to milligram quantities and confirm their biological activity. We use this method to solubilize monomeric or multimeric components of multi-protein complexes and demonstrate its efficacy to reconstitute large cellular machines. This protocol works equally well on cytosolic, nuclear and membrane proteins and can be easily adapted to a high throughput format.
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, USA.
authors.forename: Filippo
authors.initials: F
authors.lastname: Pullara
authors.forename: Jennifer
authors.initials: J
authors.lastname: Guerrero-Santoro
authors.forename: Monica
authors.initials: M
authors.lastname: Calero
authors.forename: Qiangmin
authors.initials: Q
authors.lastname: Zhang
authors.forename: Ye
authors.initials: Y
authors.lastname: Peng
authors.forename: Henrik
authors.initials: H
authors.lastname: Spåhr
authors.forename: Guy L
authors.initials: GL
authors.lastname: Kornberg
authors.forename: Antonella
authors.initials: A
authors.lastname: Cusimano
authors.forename: Hilary P
authors.initials: HP
authors.lastname: Stevenson
authors.forename: Hugo
authors.initials: H
authors.lastname: Santamaria-Suarez
authors.forename: Shelley L
authors.initials: SL
authors.lastname: Reynolds
authors.forename: Ian S
authors.initials: IS
authors.lastname: Brown
authors.forename: Satdarshan P S
authors.initials: SP
authors.lastname: Monga
authors.forename: Bennett
authors.initials: B
authors.lastname: Van Houten
authors.forename: Vesna
authors.initials: V
authors.lastname: Rapić-Otrin
authors.forename: Guillermo
authors.initials: G
authors.lastname: Calero
authors.forename: Arthur S
authors.initials: AS
authors.lastname: Levine
mesh_terms: D001709:Biotechnology / Q000379:methods*; D003902:Detergents / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae; D061987:Sf9 Cells; D012995:Solubility
chemical_list: D003902:Detergents; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D011994:Recombinant Proteins