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 . A method for producing a nanolipoprotein particle in a single reaction, the nanolipoprotein particle comprising a scaffold protein and a membrane forming lipid, the method comprising:
 providing a polynucleotide encoding for the scaffold protein; and   translating the polynucleotide to produce the scaffold protein via an in vitro cell-free translation system, in presence of the membrane forming lipid, thus producing a nanolipoprotein having a discoidal structure.   
     
     
         2 . The method of  claim 1 , wherein the target protein is a membrane protein and the membrane forming lipid is selected from the group consisting of lipids phospholipids, sphingolipids, glycolipids, ether lipids, sterols and alkylphosphocholins. 
     
     
         3 . The method of  claim 1 , wherein the target protein is selected from the group consisting of a protein coupled receptor (GPCR), an ion channel protein (IC) and a small multidrug resistance transporter (SMR). 
     
     
         4 . The method of  claim 1 , wherein the polynucleotide is an engineered polynucleotide encoding for a chimeric product. 
     
     
         5 . The method of  claim 1 , wherein the target protein is selected from the group consisting of bacteriorhodopsin, V2R, CRF, ETB, MC5R, NTR1, 5HT1A, H2, M1,herg, α1AR, β1AR, OP1R,β2AR and M2. 
     
     
         6 . A method for producing a nanolipoprotein particle, the nanolipoprotein particle comprising a scaffold protein, a membrane forming lipid and a target protein, the method comprising:
 providing a polynucleotide encoding for the target protein; and   translating the polynucleotide to produce the target protein via in vitro cell free translation system, in presence of the scaffold protein and the membrane forming lipid.   
     
     
         7 . The method of  claim 6 , 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. 
     
     
         8 . The method of  claim 6 , wherein the target protein is selected from the group consisting of a protein coupled receptor (GPCR), an ion channel protein (IC) and a small multidrug resistance transporter (SMR). 
     
     
         9 . The method of  claim 6 , wherein the polynucleotide is an engineered polynucleotide encoding for a chimeric product. 
     
     
         10 . The method of  claim 6 , wherein the target protein is selected from the group consisting of bachteriorhodopsin, V2R, CRF, ETB, MC 5 R, NTR1, 5HT1A, H2, M1,herg, α1AR, β1AR, OP1R,β2AR and M2. 
     
     
         11 . A method for producing a nanolipoprotein particle, the nanolipoprotein particle comprising a scaffold protein, a membrane forming lipid and a target protein, the method comprising:
 providing a first polynucleotide encoding for the scaffold protein;   providing a second polynucleotide encoding for the target protein; and   translating the first polynucleotide and the second polynucleotide in an in vitro cell free translation system, in presence of the membrane forming lipid.   
     
     
         12 . The method of  claim 11 , wherein contacting the first and second polynucleotide with the membrane forming lipid and the scaffold protein is performed simultaneously in a single reaction mixture. 
     
     
         13 . The method of  claim 11 , 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. 
     
     
         14 . The method of  claim 11 , wherein the target protein is selected from the group consisting of a protein coupled receptor (GPCR), an ion channel protein (IC) and a small multidrug resistance transporter (SMR). 
     
     
         15 . The method of  claim 11 , wherein at least one of the first and the second polynucleotide is an engineered polynucleotide encoding for a chimeric product. 
     
     
         16 . The method of  claim 11 , wherein the target protein is selected from the group consisting of bacteriorhodopsin, V2R, CRF, ETB, MC5R, NTR1, 5HT1A, H2, M1,herg, α1AR, β1AR, OP1R,β2AR and M2. 
     
     
         17 . A system 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 system 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.   
     
     
         18 . The system of  claim 17 , wherein the polynucleotide is engineered to provide a chimeric product. 
     
     
         19 . The system 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 system 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 system of  claim 17 , wherein the target protein is selected from the group consisting of bacteriorodhopsin, V2R, CRF, ETB, MC5R, NTR1, 5HT1A, H2, M1,herg, α1AR, β1AR, OP1R,β2AR and M2. 
     
     
         22 . The system of  claim 17 , further comprising a labeled molecule that specifically binds to the target protein, the labeled molecule providing a labeling signal. 
     
     
         23 . The system of  claim 22 , wherein the labeled molecule is selected from the group consisting of radioactive isotopes, chemioluminescent dyes, fluorophores, chromophores, enzymes, enzymes substrates, enzyme cofactor, enzyme inhibitors, dyes, metal ions, nanoparticles, metal sos and ligands. 
     
     
         24 . A system 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 system 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.   
     
     
         25 . The system of  claim 24 , wherein the polynucleotide is engineered to provide a chimeric product. 
     
     
         26 . The system 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 system 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 system of  claim 24 , wherein the target protein is selected from the group consisting of bacteriorodhopsin, V2R, CRF, ETB, MC5R, NTR1, 5HT1A, H2, M1,herg, α1AR, β1AR, OP1R,β2AR and M2. 
     
     
         29 . The system of  claim 28 , further comprising a labeled molecule that specifically binds to the target protein, the labeled molecule providing a labeling signal. 
     
     
         30 . The system of  claim 29 , wherein the labeled molecule is selected from the group consisting of radioactive isotopes, chemioluminescent dyes, fluorophores, chromophores, enzymes, enzymes substrates, enzyme cofactor, enzyme inhibitors, dyes, metal ions, nanoparticles, metal sos and ligands. 
     
     
         31 . A system 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 system 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.   
     
     
         32 . The system of  claim 31 , wherein at least one of the first and the second polynucleotide is engineered to provide a chimeric product. 
     
     
         33 . The system 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 system 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 system of  claim 31 , wherein the target protein is selected from the group consisting of bacteriorodhopsin, V2R, CRF, ETB, MC5R, NTR1, 5HT1A, H2, M1,herg, α1AR, β1AR, OP1R,β2AR and M2. 
     
     
         36 . The system of  claim 31 , further comprising a labeled molecule that specifically binds to the target protein, the labeled molecule providing a labeling signal. 
     
     
         37 . The system of  claim 36 , wherein the labeled molecule is selected from the group consisting of radioactive isotopes, chemioluminescent dyes, fluorophores, chromophores, enzymes, enzymes substrates, enzyme cofactor, enzyme inhibitors, dyes, metal ions, nanoparticles, metal sos and ligands. 
     
     
         38 . A method for producing a nanolipoprotein particle, the nanolipoprotein particle comprising a scaffold protein and a membrane forming lipid, the method comprising:
 contacting the scaffold protein and the membrane forming lipid for a time and under conditions to allow self assembly of said scaffold protein and said membrane forming lipid,   the scaffold protein and the membrane forming lipid bing contacted in a mass ratio ranging from 3:1 to 6:1.   
     
     
         39 . A method for producing a nanolipoprotein particle of a predefined size, the predefined size being from 10 to 60 nm, the nanolipoprotein particle comprising a scaffold protein and a membrane forming lipid, the method comprising:
 contacting the scaffold protein and the membrane forming lipid for a time and under conditions to allow self assembly of said scaffold protein and said membrane forming lipid,   the scaffold protein and the membrane forming lipid being contact at a mass ratio of scaffold protein to membrane forming lipid ranging from 3:1 to 4:1.   
     
     
         40 . A method for producing a nanolipoprotein particle, the nanolipoprotein particle comprising a scaffold protein, a membrane forming lipid and a target protein, the nanolipoprotein particle configured to include a predetermined target protein, the method comprising
 contacting the scaffold protein, the membrane forming lipid and the predetermined target protein for a time and under conditions to allow self assembly of said scaffold protein and said membrane forming lipid,   the scaffold protein, the membrane forming lipid being contact at a final mass ratio of scaffold protein:membrane forming lipid:predetermined target protein of about 4:1:6.