PMID- 27233794
OWN - NLM
STAT- MEDLINE
DCOM- 20171207
LR  - 20181113
IS  - 1573-5001 (Electronic)
IS  - 0925-2738 (Linking)
VI  - 65
IP  - 2
DP  - 2016 Jun
TI  - Cell-free expression, purification, and membrane reconstitution for NMR studies 
      of the nonstructural protein 4B from hepatitis C virus.
PG  - 87-98
LID - 10.1007/s10858-016-0040-2 [doi]
AB  - We describe the expression of the hepatitis C virus nonstructural protein 4B 
      (NS4B), which is an integral membrane protein, in a wheat germ cell-free system, 
      the subsequent purification and characterization of NS4B and its insertion into 
      proteoliposomes in amounts sufficient for multidimensional solid-state NMR 
      spectroscopy. First spectra of the isotopically [(2)H,(13)C,(15)N]-labeled 
      protein are shown to yield narrow (13)C resonance lines and a proper, 
      predominantly alpha-helical fold. Clean residue-selective leucine, isoleucine and 
      threonine-labeling is demonstrated. These results evidence the suitability of the 
      wheat germ-produced integral membrane protein NS4B for solid-state NMR. Still, 
      the proton linewidth under fast magic angle spinning is broader than expected for 
      a perfect sample and possible causes are discussed.
FAU - Fogeron, Marie-Laure
AU  - Fogeron ML
AD  - Institut de Biologie et Chimie des Proteines, Bases Moleculaires et Structurales 
      des Systemes Infectieux, Labex Ecofect, UMR 5086 CNRS, Universite de Lyon, 7 
      passage du Vercors, 69367, Lyon, France.
FAU - Jirasko, Vlastimil
AU  - Jirasko V
AD  - Physical Chemistry, ETH Zurich, 8093, Zurich, Switzerland.
AD  - Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im 
      Neuenheimer Feld 345, 69120, Heidelberg, Germany.
AD  - German Centre for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, 
      Germany.
FAU - Penzel, Susanne
AU  - Penzel S
AD  - Physical Chemistry, ETH Zurich, 8093, Zurich, Switzerland.
FAU - Paul, David
AU  - Paul D
AD  - Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im 
      Neuenheimer Feld 345, 69120, Heidelberg, Germany.
AD  - German Centre for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, 
      Germany.
FAU - Montserret, Roland
AU  - Montserret R
AD  - Institut de Biologie et Chimie des Proteines, Bases Moleculaires et Structurales 
      des Systemes Infectieux, Labex Ecofect, UMR 5086 CNRS, Universite de Lyon, 7 
      passage du Vercors, 69367, Lyon, France.
FAU - Danis, Clement
AU  - Danis C
AD  - Institut de Biologie et Chimie des Proteines, Bases Moleculaires et Structurales 
      des Systemes Infectieux, Labex Ecofect, UMR 5086 CNRS, Universite de Lyon, 7 
      passage du Vercors, 69367, Lyon, France.
FAU - Lacabanne, Denis
AU  - Lacabanne D
AD  - Institut de Biologie et Chimie des Proteines, Bases Moleculaires et Structurales 
      des Systemes Infectieux, Labex Ecofect, UMR 5086 CNRS, Universite de Lyon, 7 
      passage du Vercors, 69367, Lyon, France.
FAU - Badillo, Aurelie
AU  - Badillo A
AD  - Institut de Biologie et Chimie des Proteines, Bases Moleculaires et Structurales 
      des Systemes Infectieux, Labex Ecofect, UMR 5086 CNRS, Universite de Lyon, 7 
      passage du Vercors, 69367, Lyon, France.
AD  - Recombinant Protein Unit, RD-Biotech, 3 rue Henri Baigue, 25000, Besancon, 
      France.
FAU - Gouttenoire, Jerome
AU  - Gouttenoire J
AD  - Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire 
      Vaudois, University of Lausanne, 1011, Lausanne, Switzerland.
FAU - Moradpour, Darius
AU  - Moradpour D
AD  - Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire 
      Vaudois, University of Lausanne, 1011, Lausanne, Switzerland.
FAU - Bartenschlager, Ralf
AU  - Bartenschlager R
AD  - Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im 
      Neuenheimer Feld 345, 69120, Heidelberg, Germany.
AD  - German Centre for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, 
      Germany.
FAU - Penin, Francois
AU  - Penin F
AD  - Institut de Biologie et Chimie des Proteines, Bases Moleculaires et Structurales 
      des Systemes Infectieux, Labex Ecofect, UMR 5086 CNRS, Universite de Lyon, 7 
      passage du Vercors, 69367, Lyon, France.
FAU - Meier, Beat H
AU  - Meier BH
AUID- ORCID: 0000-0002-9107-4464
AD  - Physical Chemistry, ETH Zurich, 8093, Zurich, Switzerland. beme@ethz.ch.
FAU - Bockmann, Anja
AU  - Bockmann A
AD  - Institut de Biologie et Chimie des Proteines, Bases Moleculaires et Structurales 
      des Systemes Infectieux, Labex Ecofect, UMR 5086 CNRS, Universite de Lyon, 7 
      passage du Vercors, 69367, Lyon, France. a.bockmann@ibcp.fr.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20160527
PL  - Netherlands
TA  - J Biomol NMR
JT  - Journal of biomolecular NMR
JID - 9110829
RN  - 0 (Membrane Proteins)
RN  - 0 (NS4B protein, flavivirus)
RN  - 0 (Proteolipids)
RN  - 0 (Viral Nonstructural Proteins)
RN  - 0 (proteoliposomes)
SB  - IM
MH  - Amino Acid Sequence
MH  - Carbon-13 Magnetic Resonance Spectroscopy
MH  - Circular Dichroism
MH  - *Gene Expression
MH  - Humans
MH  - *Magnetic Resonance Spectroscopy/methods
MH  - Membrane Proteins/*chemistry
MH  - *Nuclear Magnetic Resonance, Biomolecular/methods
MH  - Protein Domains
MH  - Proteolipids/chemistry
MH  - Viral Nonstructural Proteins/*chemistry/*genetics/*isolation & purification
OTO - NOTNLM
OT  - Cell-free protein expression
OT  - Integral membrane protein
OT  - Isotope labeling
OT  - Lipid reconstitution
OT  - NS4B
OT  - Solid-state NMR
EDAT- 2016/05/29 06:00
MHDA- 2017/12/08 06:00
CRDT- 2016/05/29 06:00
PHST- 2016/03/08 00:00 [received]
PHST- 2016/05/21 00:00 [accepted]
PHST- 2016/05/29 06:00 [entrez]
PHST- 2016/05/29 06:00 [pubmed]
PHST- 2017/12/08 06:00 [medline]
AID - 10.1007/s10858-016-0040-2 [pii]
AID - 10.1007/s10858-016-0040-2 [doi]
PST - ppublish
SO  - J Biomol NMR. 2016 Jun;65(2):87-98. doi: 10.1007/s10858-016-0040-2. Epub 2016 May 
      27.