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

This application claims priority to U.S. Provisional Application entitled “Cell-free Self Assembly of Nano-lipoprotein Particles as a Platform for Co-expressed Membrane Proteins” Ser. No. 60/928,579, filed on May 9, 2007 Docket No. IL-11841, and to U.S. Provisional Application entitled “Monitoring IVT or Cell-free Membrane Protein Expressions, Folding and Functional Using In Situ Expression of Bacteriorhodopsin as an Internal Colorimetric” Ser. No. 60/928,573 filed on May 9, 2007 Docket No. IL-11842, the disclosures of which are incorporated herein by reference in their entirety. This application may also be related to U.S. application entitled “Methods and Systems for Producing Nanolipoprotein Particle” filed on the same day of the present application with Docket No. P197-US, the disclosure of which is also incorporated herein by reference in its entirety.

The United States Government has rights in this invention pursuant to Contract No. DE-AC52-07NA27344 between the U.S. Department of Energy and Lawrence Livermore National Security, LLC, for the operation of Lawrence Livermore National Security.”

The present disclosure relates to membranes and membrane associated proteins and to complexes mimicking said membranes and membrane associated proteins.

Membrane-associated proteins and protein complexes account for—30% or more of the cellular proteins. Membrane proteins are held within a bilayer structure. The basic membrane bilayer construct consists of two opposing layers of amphiphilic molecules know as phospholipids; each molecule has a hydrophilic moiety, i.e., a polar phosphate group/derivative, and a hydrophobic moiety, i.e., a long hydrocarbon chain. These molecules self-assemble in a biological (largely aqueous) environment according to thermodynamics associated with water exclusion or hydrophobic association.

In order to facilitate the myriad functions of biological membranes including the passage of nutrients, signaling molecules and other molecules into and out of the cell, membrane proteins are arrayed in the bilayer structure as depicted below. Note that some proteins span the bilayer, others are anchored within the bilayer, and still others organize “peripheral” proteins into complexes. Many membrane bound protein complexes mediate essential cellular processes e.g. signal transduction, transport, recognition, and cell-cell communication. In general, this class of proteins is challenging to study because of their insolubility and tendency to aggregate when removed from their protein lipid bilayer environment.

Membrane proteins are optimally folded and functional when in a lipid bilayer, but standard protein purification methods often remove lipids, invariably altering protein conformation and function.

Furthermore, also non-membrane proteins (i.e. proteins that do not exercise a biological activity in connection with a location on a membrane) may still be desirably associated with a membrane for the purpose of solubilization and/or transporting and delivering to a cell.

To overcome these problems, fully functional integral membrane proteins and additional proteins can be assembled in lipid/protein-based particulate structures called nanolipoprotein particles (NLPs) usually comprising membrane forming lipids and apolipoproteins.

NLP assembly and function usually involves the association of specific apolipoprotein and lipid molecules leading to formation of proteolipid complexes; the latter are used to transport a diverse array of lipid molecules within organisms.

NLPs made in the presence of a solubilized membrane protein (target) result in a membrane protein NLP construct Accordingly, NLP assembly can also be used for stabilization and characterization of membrane proteins.

Provided herein, are methods and systems for monitoring production of a target protein in a NLP nanostructure. In particular, the methods and systems herein disclosed allow monitoring of synthesis, correct folding and incorporation of the target protein in a NLP, following assembly of the NLP, which in some embodiments can also occur in a single reaction.

According to a first aspect, a method for monitoring production of a target protein in a nanolipoprotein particle is disclosed. The nanolipoprotein particle comprises the target protein a membrane forming lipid and a scaffold protein. The target protein is capable of assuming a target protein active form and a target protein inactive form. The method comprises: 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. The method further comprises 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. The method also comprises detecting the indicator protein detectable activity from the nanolipoprotein particle.

According to a second aspect, a method for monitoring production of a target protein in a nanolipoprotein particle is disclosed. The nanolipoprotein particle comprises the target protein a membrane forming lipid and a scaffold protein. The target protein is capable of assuming a target protein active form and a target protein inactive form. The method comprises: providing a first polynucleotide encoding for the target protein; providing a second polynucleotide encoding for the 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. The method further comprises: 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. The method also comprises: detecting the indicator protein detectable activity from the nanolipoprotein particle.

According to a third aspect, a method for monitoring production of a target protein in a nanolipoprotein particle is disclosed. The nanolipoprotein particle comprises the target protein a membrane forming lipid and a scaffold protein. The target protein is capable of assuming a target protein active form and a target protein inactive form. The method comprises: providing a first polynucleotide encoding for the target protein; providing a second polynucleotide encoding for the 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 providing a third polynucleotide encoding for the scaffold protein. The method further comprises: 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. The method also comprises: detecting the indicator protein detectable activity from the nanolipoprotein particle.