Methods and systems for producing nanolipoprotein particles

Abstract

Provided herein are methods and systems for the production of a nanolipoprotein particle (NLP) that includes a scaffold protein a membrane forming lipid and optionally a target protein. At least one of the scaffold protein and target protein can be provided through an IVT system. The membrane forming lipid, scaffold protein and optionally the target protein can be assembled for a time and under conditions that allow obtaining high yield NLPs, NPLs with an increased solubility, an NLP of a controlled size, and/or an NLP having a size predetermined to include a pre-selected target protein.

1 .- 16 . (canceled) 
     
     
         17 . A combination for providing a nanolipoprotein particle, the nanolipoprotein particle comprising a scaffold protein and a membrane forming lipid, the system comprising:
 the membrane forming lipid; and   a polynucleotide encoding for the scaffold protein,   
       the combination configured to be operated in connection with an in vitro cell free translation system for translation of the polynucleotide in presence of the membrane forming lipid and in the absence of detergent, to provide the nanolipoprotein particle in which a discoidal membrane lipid bilayer formed by the membrane forming lipid is stabilized by the scaffold protein. 
     
     
         18 . The combination of  claim 17 , wherein the polynucleotide is an engineered polynucleotide encoding a chimeric product. 
     
     
         19 . The combination of  claim 17 , wherein the target protein is a membrane protein and the membrane forming lipid is selected from the group consisting of phospholipids, sphingolipids, glycolipids, ether lipids, sterols and alkylphosphocholins. 
     
     
         20 . The combination of  claim 17 , wherein the target protein is selected from the group consisting of a protein coupled receptor (GPCR), an ion channel protein (IC) or a small multidrug resistance transporter (SMR). 
     
     
         21 . The combination of  claim 17 , wherein the target protein is selected from the group consisting of bacteriorhodopsin, vasopressin type 2 receptor (V2R), corticotropin releasing hormone receptor 1 (CRF), endothelin receptor type B (ETB), melanocortin 5 receptor (MC5R), neurotensin receptor 1 (NTR1), 5-hydroxytryptamine receptor 1 A (5HT1A), histamine H2 receptor (H2), muscarinic acetylcholine receptor M1 (M1), hERG channel protein (hERG), α1-adrenergic receptor (α1AR), β1-adrenergic receptor (β1AR), opioid receptor type 1 (OP1R), β2-adrenergic receptor (β2AR) and muscarinic acetylcholine receptor M2 (M2). 
     
     
         22 . The combination of  claim 17 , further comprising a labeled molecule that specifically binds to the target protein, the labeled molecule providing a labeling signal. 
     
     
         23 . The combination of  claim 22 , wherein the labeled molecule is selected from the group consisting of radioactive isotopes, chemiluminescent dyes, fluorophores, chromophores, enzymes, enzymes substrates, enzyme cofactor, enzyme inhibitors, dyes, metal ions, nanoparticles, metal sos and ligands. 
     
     
         24 . A combination for producing a nanolipoprotein particle, the nanolipoprotein particle comprising a scaffold protein, a membrane forming lipid and a target protein, the system comprising
 at least one of the membrane forming lipid and the scaffold protein; and   a polynucleotide encoding for the target protein,   
       the combination configured to be operated in connection with an in vitro cell free translation system for the translation of the polynucleotide in presence of the membrane forming lipid and of the scaffold protein and in absence of detergent to provide the target protein within a discoidal membrane lipid bilayer formed by the membrane forming lipid and stabilized by the scaffold protein, the membrane lipid bilayer attaching the target protein through interaction of a hydrophobic region of the target protein with the membrane lipid bilayer. 
     
     
         25 . The combination of  claim 24 , wherein the polynucleotide is engineered to provide a chimeric product. 
     
     
         26 . The combination of  claim 24 , wherein the target protein is a membrane protein and the membrane forming lipid is selected from the group consisting of phospholipids, sphingolipids, glycolipids, ether lipids, sterols and alkylphosphocholins. 
     
     
         27 . The combination of  claim 24 , wherein the target protein is selected from the group consisting of a protein coupled receptor (GPCR), an ion channel protein (IC) or a small multidrug resistance transporter (SMR). 
     
     
         28 . The combination of  claim 24 , wherein the target protein is selected from the group consisting of bacteriorhodopsin, vasopressin type 2 receptor (V2R), corticotropin releasing hormone receptor 1 (CRF), endothelin receptor type B (ETB), melanocortin 5 receptor (MC5R), neurotensin receptor 1 (NTR1), 5-hydroxytryptamine receptor 1 A (5HT1A), histamine H2 receptor (H2), muscarinic acetylcholine receptor M1 (M1), hERG channel protein (hERG), α1-adrenergic receptor (α1AR), β1-adrenergic receptor (β1AR), opioid receptor type 1 (OP1R), β2-adrenergic receptor (β2AR) and muscarinic acetylcholine receptor M2 (M2). 
     
     
         29 . The combination of  claim 28 , further comprising a labeled molecule that specifically binds to the target protein, the labeled molecule providing a labeling signal. 
     
     
         30 . The combination of  claim 29 , wherein the labeled molecule is selected from the group consisting of radioactive isotopes, chemiluminescent dyes, fluorophores, chromophores, enzymes, enzymes substrates, enzyme cofactor, enzyme inhibitors, dyes, metal ions, nanoparticles, metal sos and ligands. 
     
     
         31 . A combination for producing a nanolipoprotein particle, the nanolipoprotein particle comprising a scaffold protein, a membrane forming lipid and a target protein, the system comprising:
 a first polynucleotide encoding for the scaffold protein; and   a second polynucleotide encoding for the target protein,   
       the combination configured to be operated in connection with an in vitro cell free translation system for translation of the first and second polynucleotide in presence of the membrane forming lipid and in absence of detergent, the target protein within a discoidal membrane lipid bilayer formed by the membrane forming lipid and stabilized by the scaffold protein, the membrane lipid bilayer attaching the target protein through interaction of a hydrophobic region of the target protein with the membrane lipid bilayer. 
     
     
         32 . The combination of  claim 31 , wherein at least one of the first and the second polynucleotide is engineered to provide a chimeric product. 
     
     
         33 . The combination of  claim 31 , wherein the target protein is a membrane protein and the membrane forming lipid is selected from the group consisting of phospholipids, sphingolipids, glycolipids, ether lipids, sterols and alkylphosphocholins. 
     
     
         34 . The combination of  claim 30 , wherein the target protein is selected from the group consisting of a protein coupled receptor (GPCR), an ion channel protein (IC) or a small multidrug resistance transporter (SMR). 
     
     
         35 . The combination of  claim 31 , wherein the target protein is selected from the group consisting of bacteriorhodopsin, vasopressin type 2 receptor (V2R), corticotropin releasing hormone receptor 1 (CRF), endothelin receptor type B (ETB), melanocortin 5 receptor (MC5R), neurotensin receptor 1 (NTR1), 5-hydroxytryptamine receptor 1 A (5HT1A), histamine H2 receptor (H2), muscarinic acetylcholine receptor M1 (M1), hERG channel protein (hERG), α1-adrenergic receptor (α1AR), β1-adrenergic receptor (β1AR), opioid receptor type 1 (OP1R), β2-adrenergic receptor (β2AR) and muscarinic acetylcholine receptor M2 (M2). 
     
     
         36 . The combination of  claim 31 , further comprising a labeled molecule that specifically binds to the target protein, the labeled molecule providing a labeling signal. 
     
     
         37 . The combination of  claim 36 , wherein the labeled molecule is selected from the group consisting of radioactive isotopes, chemiluminescent dyes, fluorophores, chromophores, enzymes, enzymes substrates, enzyme cofactor, enzyme inhibitors, dyes, metal ions, nanoparticles, metal sos and ligands. 
     
     
         38 .- 40 . (canceled) 
     
     
         41 . The combination of  claim 17 , further comprising the cell-free expression system. 
     
     
         42 . The combination of  claim 17 , wherein the combination is a kit of parts. 
     
     
         43 . The combination of  claim 17 , wherein the polynucleotide is a DNA polynucleotide. 
     
     
         44 . The combination of  claim 24 , further comprising the cell-free expression system. 
     
     
         45 . The combination of  claim 24 , wherein the combination is a kit of parts. 
     
     
         46 . The combination of  claim 24 , wherein the polynucleotide is a DNA polynucleotide. 
     
     
         47 . The combination of  claim 31 , further comprising the cell-free expression system. 
     
     
         48 . The combination of  claim 31 , wherein the combination is a kit of parts. 
     
     
         49 . The combination of  claim 31 , wherein the polynucleotide is a DNA polynucleotide.