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Cell-free Assembly of NanoLipoprotein Particles

DConT2

Laboratory: Lawrence Livermore National Laboratory

Background: Membrane-associated proteins and protein complexes account for roughly one-third of the cellular proteins. These protein complexes mediate essential cellular processes such as signal transduction, transport, recognition, and cell-to-cell communication. This class of proteins is difficult to study because of their insolubility and tendency to aggregate when removed from their protein-phospholipid lipid bilayer environment. Also, over-expression of membrane proteins in vivo often results in cell toxicity, protein aggregation, mis-folding and low yield.

Description: LLNL has developed a novel process of production, isolation, characterization, and functional reconstitution of membrane-associated proteins in a single step. In addition, LLNL has developed a colorimetric assay that indicates production, correct folding, and incorporation of bR into soluble nanolipoprotein particles (NLPs).

LLNL has developed an approach for the formation of NLP/membrane protein complexes by simultaneous co-expression of both apolipoprotein and target membrane protein in a cell-free protein synthesis system. This approach involves cell-free transcription/translation technology adapted to co-express both apolipoproteins and a target membrane protein. It is carried out in a single reaction chamber with cell extract, buffer, phospholipids, detergents and the like to facilitate stabilization; the entire process can be complete in a few hours.

Advantages:

  • Cell-free protein synthesis technology allows simultaneous expression of apolipoproteins and membrane proteins, leading to self-assembly of nanolipoprotein particles containing soluble and functional proteins within a stable membrane mimetic
  • Cell-free method of simultaneous co-expression of both apolipoprotein and target membrane can be achieved in a single step
  • Process can be completed in a few hours
  • Increase yields of stable, soluble and functional membrane proteins for downstream characterization
  • Membrane proteins are expressed rather than purified from cellular sources
  • Rapid production of novel functional membrane proteins that are difficult to obtain from native systems
  • Process screening of parameters for evaluation of production of membrane proteins
  • Medical diagnostics, therapeutics, biofuels

Development Status: LLNL has used cell-free expression for single-step production and refolding of the membrane protein bacteriorhodopsin. This unique approach has been demonstrated by the co-expression of a truncated apolipoprotein and the bacteriorhodopsin gene, which resulted in a functionally active seven trans-membrane helix spanning bacteriorhodopsin protein containing co-factor retinal. In addition, LLNL has developed a colorimetric assay that indicates production, correct folding, and incorporation of bacteriorhodopsin into soluble nanolipoprotein particles.

Opportunity: LLNL has filed provisional patent applications on these technologies. Licensing opportunities may be available.

Contact: Annemarie Meike

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