Methods and systems for monitoring production of a target protein in a nanolipoprotein particle

Abstract

Provided herein are methods and systems for the monitoring production of a target protein in of a nanolipoprotein particle (NLP) that also includes a scaffold protein and a membrane forming lipid. The target protein is capable of assuming an active form and an inactive form. Monitoring is performed by an indicator protein that is capable of assuming an active form and an inactive form, the active form associated with a detectable activity of the indicator protein, the detectable activity further associated with the active form of the target protein.

1 . A method for monitoring production of a target protein in a nanolipoprotein particle,
 the nanolipoprotein particle comprising the target protein, a membrane forming lipid and a scaffold protein,   the target protein capable of assuming a target protein active form and a target protein inactive form,   the method comprising:
 providing an indicator protein, the indicator protein capable of assuming an indicator protein active form and an indicator protein inactive form, the indicator protein active form associated to an indicator protein detectable activity, the indicator protein detectable activity associated to the target protein active form; 
 contacting the indicator protein with the target protein, the membrane forming lipid and the scaffold protein for a time and under conditions to allow assembly of the indicator protein, the target protein, the membrane forming lipid and the scaffold protein in the nanolipoprotein particle; and 
 detecting the indicator protein detectable activity from the nanolipoprotein particle. 
   
     
     
         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 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 target protein is selected from the group consisting of V2R, CRF, ETB, MC5R, NTR1, 5HT1A, H2, M1, herg, α1AR, β1AR, OP1R, β2AR and M2. 
     
     
         5 . The method of  claim 1 , wherein the indicator protein is structurally related to the target protein so that the production of the indicator protein in an active form can be associated with the production of the target protein in an active form. 
     
     
         6 . The method of  claim 1  wherein the indicator protein is selected from the group consisting of GFP, GFP-fused to a membrane protein, cytochromes and dye labeled proteins. 
     
     
         7 . The method of  claim 1 , wherein the indicator protein is selected from the group consisting of sensory rhodopsin, proteorhodopsin, and phytochromes. 
     
     
         8 . The method of  claim 1 , wherein the indicator protein is bacteriorhodopsin. 
     
     
         9 . The method of  claim 1 , wherein detecting the indicator protein detectable activity is performed by
 providing a labeled molecule that specifically binds to the indicator protein the labeled molecule providing a labeling signal;   contacting the labeled molecule with the nanolipoprotein particle for a time and under condition to allow binding of the labeled molecule with the indicator protein in the nanolipoprotein particle; and   detecting the labeling signal from the labeled molecule bound to the indicator protein in the nanolipoprotein particle.   
     
     
         10 . The method of  claim 9 , 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 ions and ligands. 
     
     
         11 . A method for monitoring production of a target protein in a nanolipoprotein particle,
 the nanolipoprotein particle comprising the target protein a membrane forming lipid and a scaffold protein,   the target protein capable of assuming a target protein active form and a target protein inactive form,   the method comprising:
 providing a first polynucleotide encoding for the target protein; 
 providing a second polynucleotide encoding for an indicator protein, the indicator protein capable of assuming an indicator protein active form and an indicator protein inactive form, the indicator protein active form associated to an indicator protein detectable activity, the indicator protein detectable activity associated to the target protein active form; 
 contacting the first and second polynucleotides with the membrane forming lipid and the scaffold protein for a time and under conditions to allow assembly of the indicator protein, the target protein, the membrane forming lipid and the scaffold protein in the nanolipoprotein particle; and 
 detecting the indicator protein detectable activity from the nanolipoprotein particle. 
   
     
     
         12 . The method of  claim 11 , wherein contacting the first and second polynucleotide with the membrane forming lipid and the scaffold protein is performed 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 V2R, CRF, ETB, MC5R, NTR1, 5HT1A, H2, M1, herg, α1AR, β1AR, OP1R, β2AR and M2. 
     
     
         17 . The method of  claim 11 , wherein the indicator protein is structurally related to the target protein of interest so that the production of the indicator protein in an active form can be associated with the production of the target protein in an active form. 
     
     
         18 . The method of  claim 11  wherein the indicator protein is selected from the group consisting of GFP, GFP-fused to a membrane protein, cytochromes and dye labeled proteins. 
     
     
         19 . The method of  claim 11 , wherein the indicator protein is selected from the group consisting of sensory rhodopsin, proteorhodopsin, and phytochromes. 
     
     
         20 . The method of  claim 11 , wherein the indicator protein is bacteriorhodopsin. 
     
     
         21 . The method of  claim 11 , wherein detecting the indicator protein detectable activity is performed by
 providing a labeled molecule that specifically binds to the indicator protein the labeled molecule providing a labeling signal;   contacting the labeled molecule with the nanolipoprotein particle for a time and under condition to allow binding of the labeled molecule with the indicator protein in the nanolipoprotein particle; and   detecting the labeling signal from the labeled molecule bound to the indicator protein in the nanolipoprotein particle.   
     
     
         22 . The method of  claim 21 , 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 ions and ligands. 
     
     
         23 . A method for monitoring production of a target protein in a nanolipoprotein particle, the nanolipoprotein particle comprising the target protein, a membrane forming lipid and a scaffold protein,
 the target protein capable of assuming a target protein active form and a target protein inactive form,   the method comprising:
 providing a first polynucleotide encoding for the target protein; 
 providing a second polynucleotide encoding for an indicator protein, the indicator protein capable of assuming an indicator protein active form and an indicator protein inactive form, the indicator protein active form associated to an indicator protein detectable activity, the indicator protein detectable activity associated to the target protein active form; 
 providing a third polynucleotide encoding for the scaffold protein; 
 contacting the first, second and third polynucleotides with the membrane forming lipid and the scaffold protein for a time and under conditions to allow assembly of the indicator protein, the target protein, the membrane forming lipid and the scaffold protein in the nanolipoprotein particle; and 
 detecting the indicator protein detectable activity from the nanolipoprotein particle. 
   
     
     
         24 . The method of  claim 23 , wherein contacting the first, second and third polynucleotided with the membrane forming lipid and the scaffold protein is performed in a single reaction mixture. 
     
     
         25 . The method of  claim 23 , 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. 
     
     
         26 . The method of  claim 23 , 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). 
     
     
         27 . The method of  claim 23 , wherein at least one of the first, the second polynucleotide and the third polynucleotide is an engineered polynucleotide encoding for a chimeric product. 
     
     
         28 . The method of  claim 23 , wherein the target protein is selected from the group consisting of V2R, CRF, ETB, MC5R, NTR1, 5HT1A, H2, M1, herg, α1AR, β1AR, OP1R, β2AR and M2. 
     
     
         29 . The method of  claim 23 , wherein the indicator protein is structurally related to the target protein of interest so that the production of the indicator protein in an active form can be associated with the production of the target protein in an active form. 
     
     
         30 . The method of  claim 23  wherein the indicator protein is selected from the group consisting of GFP, GFP-fused to a membrane protein, cytochromes and dye labeled proteins. 
     
     
         31 . The method of  claim 23 , wherein the indicator protein is selected from the group consisting of sensory rhodopsin, proteorhodopsin, and phytochromes. 
     
     
         32 . The method of  claim 23 , wherein the indicator protein is bacteriorhodopsin. 
     
     
         33 . The method of  claim 23 , wherein detecting the indicator protein detectable activity is performed by
 providing a labeled molecule that specifically binds to the indicator protein the labeled molecule providing a labeling signal;   contacting the labeled molecule with the nanolipoprotein particle for a time and under condition to allow binding of the labeled molecule with the indicator protein in the nanolipoprotein particle; and   detecting the labeling signal from the labeled molecule bound to the indicator protein in the nanolipoprotein particle.   
     
     
         34 . The method of  claim 33 , 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 ions and ligands. 
     
     
         35 . A system for monitoring production of a target protein in a nanolipoprotein particle,
 the nanolipoprotein particle comprising the target protein, a membrane forming lipid and a scaffold protein,   the target protein capable of assuming a target protein active form and a target protein inactive form,   the system comprising:
 an indicator protein capable of assuming an indicator protein active form and an indicator protein inactive form, the indicator protein active form associated to an indicator protein detectable activity, the indicator protein detectable activity associated to the target protein active form, and 
 at least one of the target protein, the membrane forming lipid and the scaffold protein. 
   
     
     
         36 . The system of  claim 35 , 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. 
     
     
         37 . The system of  claim 35 , 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). 
     
     
         38 . The system of  claim 35 , wherein the target protein is selected from the group consisting of V2R, CRF, ETB, MC5R, NTR1, 5HT1A, H2, M1, herg, α1AR, β1AR, OP1R, β2AR and M2. 
     
     
         39 . The system of  claim 35 , wherein the indicator protein is structurally related to the target protein of interest so that the production of the indicator protein in an active form can be associated with the production of the target protein in an active form. 
     
     
         40 . The system of  claim 35 , wherein the indicator protein is selected from the group consisting of GFP, GFP-fused to a membrane protein, cytochromes and dye labeled proteins. 
     
     
         41 . The system of  claim 35 , wherein the indicator protein is selected from the group consisting of sensory rhodopsin, proteorhodopsin, and phytochromes. 
     
     
         42 . The system of  claim 35 , wherein the indicator protein is bacteriorhodopsin. 
     
     
         43 . The system of  claim 35 , further comprising a labeled molecule that specifically binds to the indicator protein, the labeled molecule providing a labeling signal. 
     
     
         44 . The system of  claim 43 , 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 ions and ligands. 
     
     
         45 . A system for monitoring production of a target protein in a nanolipoprotein particle,
 the nanolipoprotein particle comprising the target protein, a membrane forming lipid and a scaffold protein,   the target protein capable of assuming a target protein active form and a target protein inactive form,   the system comprising:
 a first polynucleotide encoding for the target protein; and 
 a second polynucleotide encoding for an indicator protein, the indicator protein capable of assuming an indicator protein active form and an indicator protein inactive form, the indicator protein active form associated to an indicator protein detectable activity, the indicator protein detectable activity associated to the target protein active form. 
   
     
     
         46 . The system of  claim 45 , 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. 
     
     
         47 . The system of  claim 45 , 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). 
     
     
         48 . The system of  claim 45 , wherein the target protein is selected from the group consisting of V2R, CRF, ETB, MC5R, NTR1, 5HT1A, H2, M1, herg, α1AR, β1AR, OP1R, β2AR and M2. 
     
     
         49 . The system of  claim 45 , wherein the indicator protein is structurally related to the target protein of interest so that the production of the indicator protein in an active form can be associated with the production of the target protein in an active form. 
     
     
         50 . The system of  claim 45 , wherein the indicator protein is selected from the group consisting of GFP, GFP-fused to a membrane protein, cytochromes and dye labeled proteins. 
     
     
         51 . The system of  claim 45 , wherein the indicator protein is selected from the group consisting of sensory rhodopsin, proteorhodopsin, and phytochromes. 
     
     
         52 . The system of  claim 45 , wherein the indicator protein is bacteriorhodopsin. 
     
     
         53 . The system of  claim 45 , further comprising a labeled molecule that specifically binds to the indicator protein, the labeled molecule providing a labeling signal. 
     
     
         54 . The system of  claim 53 , 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 ions and ligands. 
     
     
         55 . A system for monitoring production of a target protein in a nanolipoprotein particle,
 the nanolipoprotein particle comprising the target protein, a membrane forming lipid and a scaffold protein,   the target protein capable of assuming a target protein active form and a target protein inactive form,   the system comprising:
 a first polynucleotide encoding for the target protein; 
 a second polynucleotide encoding for an indicator protein, the indicator protein capable of assuming an indicator protein active form and an indicator protein inactive form, the indicator protein active form associated to an indicator protein detectable activity, the indicator protein detectable activity associated to the target protein active form; and 
 a third polynucleotide encoding for the scaffold protein. 
   
     
     
         56 . The system of  claim 55 , 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. 
     
     
         57 . The system of  claim 55 , 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). 
     
     
         58 . The system of  claim 55 , wherein the target protein is selected from the group consisting of V2R, CRF, ETB, MC5R, NTR1, 5HT1A, H2, M1, herg, α1AR, β1AR, OP1R, β2AR and M2. 
     
     
         59 . The system of  claim 55 , wherein the indicator protein is structurally related to the target protein of interest so that the production of the indicator protein in an active form can be associated with the production of the target protein in an active form. 
     
     
         60 . The system of  claim 55 , wherein the indicator protein is selected from the group consisting of GFP, GFP-fused to a membrane protein, cytochromes and dye labeled proteins. 
     
     
         61 . The system of  claim 55 , wherein the indicator protein is selected from the group consisting of sensory rhodopsin, proteorhodopsin, and phytochromes. 
     
     
         62 . The system of  claim 55 , wherein the indicator protein is bacteriorhodopsin. 
     
     
         63 . The system of  claim 55 , further comprising a labeled molecule that specifically binds to the indicator protein, the labeled molecule providing a labeling signal. 
     
     
         64 . The system of  claim 63 , 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 ions and ligands.