Production of membrane proteins for NMR studies using the condensed single protein (cSPP) production system

  • Lili Mao
  • Yuefeng Tang
  • S. Thangminlal Vaiphei
  • Tsutomu Shimazu
  • Sung-Gun Kim
  • Rajeswari Mani
  • Elias Fakhoury
  • Eileen White
  • Gaetano T. Montelione
  • Masayori Inouye
Article

Abstract

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. Here we demonstrate that three prokaryotic inner membrane proteins, two prokaryotic outer membrane proteins, and one human virus membrane protein can be produced at very high levels, and assembled in appropriate membrane fractions. The condensed SPP (cSPP) system was used to selectively produce isotope-enriched membrane proteins for NMR studies in up to 150-fold condensed culture without affecting protein yields, providing more than 99% cost saving for isotopes. As a novel application of the cSPP system for studies of membrane proteins prior to purification 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.

Keywords

cSPP Membrane protein NMR 

Abbreviations

LMPC

1-Myristoyl-2-hydroxy-sn-glycero-3-phosphocholine

LMPG

1-Myristoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)]

LOPC

1-Oleoyl-2-hydroxysn-glycero-3- phosphocholine

LOPG

1-Oleoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)]

LPPG

1-Palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)]

βOG

n-Octyl-β-d-glucopyranoside

Zwittergent 3-16

Hexadecyl-N-sulfobetaine

cSPP

Condensed single protein production system

Supplementary material

10969_2009_9072_MOESM1_ESM.doc (38 kb)
Supplementary material 1 (DOC 39 kb)
10969_2009_9072_MOESM2_ESM.pdf (66 kb)
Supplementary material 2 (PDF 66 kb)

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Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Lili Mao
    • 1
    • 2
  • Yuefeng Tang
    • 1
    • 3
  • S. Thangminlal Vaiphei
    • 1
    • 2
  • Tsutomu Shimazu
    • 1
    • 2
  • Sung-Gun Kim
    • 1
    • 2
  • Rajeswari Mani
    • 1
    • 3
  • Elias Fakhoury
    • 1
    • 2
  • Eileen White
    • 4
    • 5
  • Gaetano T. Montelione
    • 1
    • 3
    • 6
  • Masayori Inouye
    • 1
    • 2
  1. 1.New York Center on Membrane Protein Structure, RutgersThe State University of New JerseyPiscatawayUSA
  2. 2.Center for Advanced Biotechnology and Medicine (CABM)UMDNJ-Robert Wood Johnson Medical SchoolPiscatawayUSA
  3. 3.Department of Molecular Biology and Biochemistry, RutgersCenter for Advanced Biotechnology and Medicine (CABM)PiscatawayUSA
  4. 4.Department of Molecular Biology and Biochemistry, Rutgers The State University of New JerseyPiscatawayUSA
  5. 5.The Cancer Institute of New JerseyNew BrunswickUSA
  6. 6.Department of BiochemistryUMDNJ-Robert Wood Johnson Medical SchoolPiscatawayUSA

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