title: Cell-free expression for nanolipoprotein particles: building a high-throughput membrane protein solubility platform.
abstract: Membrane-associated proteins and protein complexes account for approximately a third or more of the proteins in the cell (1, 2). These complexes mediate essential cellular processes; including signal transduc-tion, transport, recognition, bioenergetics and cell-cell communication. In general, membrane proteins are challenging to study because of their insolubility and tendency to aggregate when removed from their protein lipid bilayer environment. This chapter is focused on describing a novel method for producing and solubilizing membrane proteins that can be easily adapted to high-throughput expression screening. This process is based on cell-free transcription and translation technology coupled with nanolipoprotein par ticles (NLPs), which are lipid bilayers confined within a ring of amphipathic protein of defined diameter. The NLPs act as a platform for inserting, solubilizing and characterizing functional membrane proteins. NLP component proteins (apolipoproteins), as well as membrane proteins can be produced by either traditional cell-based or as discussed here, cell-free expression methodologies.
authors.affiliation: Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory, Livermore, CA, USA.
authors.forename: Jenny A
authors.initials: JA
authors.lastname: Cappuccio
authors.forename: Angela K
authors.initials: AK
authors.lastname: Hinz
authors.forename: Edward A
authors.initials: EA
authors.lastname: Kuhn
authors.forename: Julia E
authors.initials: JE
authors.lastname: Fletcher
authors.forename: Erin S
authors.initials: ES
authors.lastname: Arroyo
authors.forename: Paul T
authors.initials: PT
authors.lastname: Henderson
authors.forename: Craig D
authors.initials: CD
authors.lastname: Blanchette
authors.forename: Vickie L
authors.initials: VL
authors.lastname: Walsworth
authors.forename: Michele H
authors.initials: MH
authors.lastname: Corzett
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Law
authors.forename: Joseph B
authors.initials: JB
authors.lastname: Pesavento
authors.forename: Brent W
authors.initials: BW
authors.lastname: Segelke
authors.forename: Todd A
authors.initials: TA
authors.lastname: Sulchek
authors.forename: Brett A
authors.initials: BA
authors.lastname: Chromy
authors.forename: Federico
authors.initials: F
authors.lastname: Katzen
authors.forename: Todd
authors.initials: T
authors.lastname: Peterson
authors.forename: Graham
authors.initials: G
authors.lastname: Bench
authors.forename: Wieslaw
authors.initials: W
authors.lastname: Kudlicki
authors.forename: Paul D
authors.initials: PD
authors.lastname: Hoeprich
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Coleman
mesh_terms: D000818:Animals; D019921:Biotinylation; D002458:Cell Fractionation / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D008074:Lipoproteins / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D053758:Nanoparticles / Q000737:chemistry; D040081:Protein Array Analysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility
chemical_list: D008074:Lipoproteins; D008565:Membrane Proteins; D011994:Recombinant Proteins