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The cSPP System, A Novel System for the Production of Membrane Proteins

by
Lili Mao
B. S. Biological Sciences
School of Life Sciences, University of Science and Technology of China - 2004



Thesis Advisor: Masayori Inouye, Ph.D.
Graduate Program in Biochemistry & Molecular Biology

CABM, Room 010
Piscataway

Wednesday, September 8, 2010
2:00 p.m.


Abstract

Membrane proteins are 30 to 35% of the total proteins encoded by the genome from bacteria to human, playing a number of key functions in the cells, such as cell metabolism, signal transduction, and transport for ions and various nutrients. Thus the membrane proteins are of great interest for drug targets. Therefore, the structural studies of membrane proteins are of great importance for better understanding of the mechanisms and further drug designing. In this thesis, I introduced a new expression system, the Single Protein Production (SPP) system, for production of membrane proteins for NMR structural studies, including both solution state NMR and solid state NMR.
In the Single Protein Production (SPP) method, all E. coli cellular mRNAs are eliminated by the induction of MazF, an ACA-specific mRNA interferase. When an mRNA for a membrane protein, engineered to have no ACA sequences without altering its amino acid sequence, is induced in the MazF-induced cells, E. coli is converted into a bioreactor producing only the targeted membrane protein.
It is demonstrated here that three prokaryotic inner membrane proteins, one yeast membrane protein and an ion channel for influenza A virus can be produced in the SPP system at very high levels, and assembled in appropriate membrane fractions. In addition, the condensed Single Protein Production (cSPP) system provides a cost-effective approach for producing isotope-enriched membrane proteins which can be studied by NMR methods without the requirement of purification.
As a novel application of the SPP system for studies of membrane proteins, we also demonstrate, for the first time, fast detergent screening by microcoil NMR and well-resolved NMR spectra of several targeted integral membrane proteins obtained without purification.
I also explored the SPP system to apply for structural studies of membrane proteins by solid-state NMR. It is demonstrated that a selectively isotope-enriched protein provides a “fingerprint” spectrum of the target protein. This “fingerprint” can then be used to characterize the structural integrity of the target protein in the natural membrane environment and to compare it with the structure present in purified or reconstituted samples. In this way, membrane proteins samples used in NMR studies by solid-state or solution state methods, or for crystallization experiments and subsequent structural analysis by X-ray crystallography, can be validated if they have native structures similar to the structure of the same membrane proteins in the E. coli membrane environment.


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