Cell-free expression for nanolipoprotein particles: building a high-throughput membrane protein solubility platform.
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.
Cappuccio
Jenny A
JA
Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory, Livermore, CA, USA.
Hinz
Angela K
AK
Kuhn
Edward A
EA
Fletcher
Julia E
JE
Arroyo
Erin S
ES
Henderson
Paul T
PT
Blanchette
Craig D
CD
Walsworth
Vickie L
VL
Corzett
Michele H
MH
Law
Richard J
RJ
Pesavento
Joseph B
JB
Segelke
Brent W
BW
Sulchek
Todd A
TA
Chromy
Brett A
BA
Katzen
Federico
F
Peterson
Todd
T
Bench
Graham
G
Kudlicki
Wieslaw
W
Hoeprich
Paul D
PD
Coleman
Matthew A
MA
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
D008074:Lipoproteins; D008565:Membrane Proteins; D011994:Recombinant Proteins