A bioreactor for in-cell protein NMR.
The inside of the cell is a complex environment that is difficult to simulate when studying proteins and other molecules in vitro. We have developed a device and system that provides a controlled environment for nuclear magnetic resonance (NMR) experiments involving living cells. Our device comprises two main parts, an NMR detection region and a circulation system. The flow of medium from the bottom of the device pushes alginate encapsulated cells into the circulation chamber. In the chamber, the exchange of oxygen and nutrients occurs between the media and the encapsulated cells. When the media flow is stopped, the encapsulated cells fall back into the NMR detection region, and spectra can be acquired. We have utilized the bioreactor to study the expression of the natively disordered protein alpha-synuclein, inside Escherichia coli cells.
Sharaf
Naima G
NG
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Barnes
Christopher O
CO
Charlton
Lisa M
LM
Young
Gregory B
GB
Pielak
Gary J
GJ
D000464:Alginates; D019149:Bioreactors; D003470:Culture Media; D004867:Equipment Design; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D010100:Oxygen / Q000737:chemistry; D011138:Polytetrafluoroethylene; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D051844:alpha-Synuclein / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D000464:Alginates; D003470:Culture Media; D011506:Proteins; D011994:Recombinant Proteins; D051844:alpha-Synuclein; D011138:Polytetrafluoroethylene; D010100:Oxygen