JP2009521209A - In vitro protein synthesis system for membrane proteins comprising apolipoprotein and phospholipid-apolipoprotein particles - Google Patents

Abstract

The present invention relates to the provision of in vitro protein synthesis systems and methods for producing solubilized forms of membrane proteins. In some aspects, the invention provides a method of synthesizing a protein using an in vitro protein synthesis system that includes an apolipoprotein, thereby improving the yield of soluble protein over the absence of apolipoprotein. Apolipoproteins useful for the present invention include natural apolipoproteins (including mutated sequences of wild type apolipoproteins) and modified apolipoproteins. The apolipoprotein can be provided to the in vitro protein synthesis system in a state associated with lipid or in a state not associated with lipid. The invention also provides compositions and kits for protein synthesis in solubilized forms, the compositions and kits comprising a cell extract for protein translation and one or more apolipoprotein biomolecules.

Description

  The present invention relates generally to an in vitro protein synthesis system, and more particularly to an in vitro translation system for membrane proteins.

  This patent application is a US Provisional Patent Application No. 60/721339 (Title of Invention: “In Vitro Translation Systems for Membrane Proteins that Included Phospholipid-Protein Particles”, filed September 27, 2005), US Provisional Patent Application 60/72. (Title of Invention: “In Vitro Translation Systems for Membrane Proteins that Include Phospholipid-Protein Particles”, filed on Oct. 4, 2005, US Provisional Patent Application No. 60/815750, Celle Fre Systems Inclu ing Apolipoproteins ", filed June 21, 2006), and US Provisional Patent Application No. 60/861595, title of invention:" Cell-Free Protein Synthesis of Membrane Proteins Usage Apolipoproteins ", filed June 21, 2006). All claims are hereby incorporated by reference in their entirety.

Sequence Listing This patent application also includes a Sequence Listing, the entire disclosure of which is incorporated herein by reference.

  Strategies for treating medical conditions such as age-related disorders, autoimmune diseases and cancer rely heavily on elucidating the function of proteins. The majority of drug targets are proteins, and the majority of protein drug targets are thought to be membrane proteins. Efficiently synthesizing proteins and especially membrane proteins and securing sufficient amounts for the analysis of their structure and function is extremely important for the development of new drugs that can deal with diseases.

  The in vitro protein synthesis system enables protein to be prepared from nucleic acid templates in cell-free extracts, and facilitates efficient protein synthesis and subsequent isolation, enabling high-throughput structural and functional analysis of proteins. The effect of accelerating drug development and search can be expected.

  Unfortunately, not all proteins are synthesized in soluble form in an in vitro synthesis system. Membrane proteins are often insoluble, especially when prepared with a cell-free translation system, and in order to analyze their natural structure and activity, the proteins are solubilized in denaturing surfactants. In addition, it is necessary to try to fold the protein again. These attempts are cumbersome and often unsuccessful.

  Bayburt et al. Report that nanoscale lipid-protein particles are formed immediately when a surfactant solubilized apolipoprotein A1 (“Apo A1”) is mixed with phospholipids (see FIG. Non-patent document 1). As a result of dialysis removal of the surfactant to obtain nanoscale lipid-protein particles and structural analysis thereof, it was elucidated that a lipid bilayer surrounded by Apo A1 protein was constituted. Bayburt and Sligar describe a synthetic variant of Apo A1 (“scaffold protein”) that functions similarly to Apo A1 in the formation of lipid-protein particles in the presence of a surfactant (Non-Patent Literature). 2, Patent Documents 1 and 2, the entire disclosure is incorporated herein by reference). These researchers found that when other membrane proteins were solubilized with surfactants, they were present in the same self-assembly surfactant mix, and when further dialyzed, they were incorporated into the lipid bilayer of the nanodisc. Is elucidated.

  However, this technique for preparing solubilized forms of membrane proteins still requires further ingenuity to purify and solubilize membrane proteins prior to combining with nanodisc components in a self-assembly surfactant mix. It is said. These methods require specific proteins to be individually distinguished, time consuming, cumbersome and often require the use of radical denaturants that can affect protein function. That is, a method has been developed for the convenient expression of membrane proteins in an in vitro system, soluble, natural form, or a form that can be rapidly purified using substantially purified or simpler purification methods. There is a need to prepare a protein of

US Pat. No. 7,048,949 US Patent Application Publication No. 2005/0182243 Bayburt, T .; H. Carlson, J .; W. , And Sligar, S. G. (1998) “Restitution and Imaging of a Membrane Protein in a Nanometer—Sized Phospholipid Bilayer.” Journal of Structural Biology, 123, 37-44. Civjan, N .; R. Bayburt, T .; H. Schuler, M .; A. , And Sligar, S. G. (2003) "Direct Solubilization of Heterologously Expressed Membrane Proteins by Incorporation into Nanolipid Lipiders," BioTechniques, 356, 556.

  The present invention relates to the provision of efficient systems and methods for synthesizing proteins in a cell-free in vitro synthesis system comprising apolipoproteins (including modified apolipoproteins and variants of natural apolipoproteins). In its various aspects and embodiments, the present invention relates to the provision of an efficient system and method for synthesizing solubilized forms of membrane proteins in a cell-free system.

  One embodiment of the present invention relates to provision of a method for synthesizing a target protein in vitro. The method includes adding a nucleic acid template encoding the target protein to an in vitro protein synthesis system containing an apolipoprotein, and the in vitro protein synthesis system. And synthesizing the target protein. In some preferred embodiments, the protein of interest is synthesized in solubilized form. In some preferred embodiments, the protein of interest translated using the method of the invention is a membrane protein.

  For example, the apolipoprotein used in the method of the present invention may be any apolipoprotein, such as apolipoprotein AI, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein AV, apolipoprotein B- 100, apolipoprotein B-48, apolipoprotein C-I, apolipoprotein C-II, apolipoprotein C-III, apolipoprotein D, apolipoprotein E, apolipoprotein H, lipoprotein (a), apoliphorin I, Modified using apoliphorin II or apoliphorin III, any variant of the apolipoprotein described above, or the sequence of one or more domains of a natural apolipoprotein or sequences substantially homologous thereto. Apolipoprotein include, but are not limited to.

  Some embodiments of the present invention relate to the use of an apolipoprotein variant or modified apolipoprotein having at least 15 amino acid sequence identities having at least 15 series of amino acids of the apolipoprotein, such proteins being limited Apolipoprotein AI, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein AV, apolipoprotein B-100, apolipoprotein B-48, apolipoprotein CI, apolipoprotein C-II , Apolipoprotein C-III, apolipoprotein D, apolipoprotein E, apolipoprotein H, lipoprotein (a), apoliphorin I, apoliphorin II or apoliphorin III.

  Some embodiments of the invention relate to the use of an apolipoprotein variant or modified apolipoprotein having 90% or more amino acid sequence identity, having at least 10 series of amino acids of the helical domain of the apolipoprotein. Examples include, but are not limited to, apolipoprotein AI, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein A-V, apolipoprotein B-100, apolipoprotein B-48, apolipoprotein CI, apolipoprotein Examples include protein C-II, apolipoprotein C-III, apolipoprotein D, apolipoprotein E, apolipoprotein H, lipoprotein (a), apoliphorin I, apoliphorin II or apoliphorin III.

  The apolipoprotein added to the in vitro synthesis system may be modified by performing deletion, insertion or substitution of one or more amino acids on the amino acid sequence of the wild-type apolipoprotein. The apolipoprotein added to the in vitro synthesis system may have one or more chemical or enzymatic modifications. In some embodiments, the apolipoprotein added to the in vitro synthesis system comprises a label or tag (eg, a peptide tag).

  In some preferred embodiments, the protein of interest translated using the methods of the invention is a membrane protein, and by incubating an in vitro protein synthesis system, rather than translating the protein in the absence of apolipoprotein. A large amount of target protein is synthesized in solubilized form. In some preferred embodiments, the target protein translated using the methods of the present invention is a membrane protein, and by incubating an in vitro protein synthesis system, the target protein is translated in the absence of apolipoprotein. Also, the desired soluble protein is obtained in a higher percentage with respect to all synthesized target proteins.

  In some embodiments of the methods of the invention, after synthesis of the protein of interest in the presence of the apolipoprotein, the protein of interest is associated with the apolipoprotein. In some embodiments, the protein of interest synthesized in vitro in the presence of apolipoprotein is co-isolated with the apolipoprotein.

  In some embodiments of the methods of the invention, the apolipoprotein provided in the in vitro protein synthesis system is present as phospholipid-apolipoprotein particles. In some embodiments of the methods of the invention, the apolipoprotein in the in vitro protein synthesis system is present as phospholipid-apolipoprotein particles, and the target protein synthesized in the system is associated with the phospholipid-apolipoprotein particles. In some preferred embodiments, in an in vitro reaction involving phospholipid-apolipoprotein particles, the protein of interest synthesized can be isolated along with the phospholipid-apolipoprotein particles.

  In some embodiments of the invention, the method further comprises isolating the protein of interest from an in vitro synthesis mixture. The isolation may be performed using, for example, a peptide tag that is a part of the target protein, or may be performed using a peptide tag that is a part of the apolipoprotein obtained in the in vitro protein synthesis reaction.

  In another aspect, the present invention relates to providing an in vitro protein synthesis system comprising a cell extract and an apolipoprotein. Cell extracts containing components of the protein synthesis machinery are known in the prior art and may be derived from prokaryotic or eukaryotic cells. The apolipoprotein used in the method of the present invention may be any apolipoprotein, including, but not limited to, apolipoprotein AI, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein A described herein. -V, apolipoprotein B-100, apolipoprotein B-48, apolipoprotein CI, apolipoprotein C-II, apolipoprotein C-III, apolipoprotein D, apolipoprotein E, apolipoprotein H, lipoprotein (a) Apoliphorin I, apoliphorin II or apoliphorin III, any variant of the apolipoprotein described above, or the sequence of one or more domains of a natural apolipoprotein or substantially Apolipoprotein modified using the sequences and the like.

  The apolipoprotein obtained in the in vitro synthesis system may be a modified or derivatized apolipoprotein, and the modified or derivatized apolipoprotein has one or more chemical modifications. The apolipoprotein provided in the in vitro synthesis system may comprise a tag or label.

  In vitro protein synthesis systems preferably comprise one or more chemical energy sources for providing energy to protein synthesis. Non-limiting examples of such energy sources are nucleotides (eg ATP or GTP), glycolytic intermediates, phosphorylated compounds and enzymes that generate energy. The in vitro protein synthesis system of the present invention may further comprise free amino acids, salts, buffer compounds, enzymes, inhibitors or cofactors.

  The in vitro protein synthesis system may further comprise one or more nucleic acid templates. The nucleic acid template may be a DNA template or an RNA template, and may encode any target protein to be synthesized in vitro. A nucleic acid template present in an in vitro protein synthesis system may encode multiple proteins of interest. For example, the nucleic acid template of the IVPS system may be bound to a solid support (eg, a bead, matrix, chip, array, membrane, sheet, dish or plate).

  The in vitro protein synthesis system of the present invention may further comprise one or more surfactants or one or more lipids (eg, one or more phospholipids). In some exemplary embodiments, the in vitro synthesis system of the invention may comprise an apolipoprotein associated with one or more lipids. In some exemplary embodiments, the in vitro synthesis system of the invention comprises an apolipoprotein associated with one or more phospholipids in a phospholipid-apolipoprotein particle. In these embodiments, the protein of interest, preferably synthesized in an in vitro synthesis system, forms an aggregate with phospholipid-apolipoprotein particles. In a preferred embodiment, the protein of interest synthesized in an in vitro synthesis system can be isolated with phospholipid-apolipoprotein particles.

  In yet another aspect, the present invention relates to providing a method for synthesizing a protein in vitro, and in particular, the method comprises adding a nucleic acid construct encoding an apolipoprotein and a nucleic acid construct encoding a protein of interest to an in vitro synthesis system. Incubating an in vitro protein synthesis system to synthesize an apolipoprotein and a protein of interest. In some preferred embodiments, the protein of interest is synthesized in a solubilized form. In some preferred embodiments, the protein of interest is a membrane protein.

  In some embodiments, the apolipoprotein is provided in a first nucleic acid construct and the protein of interest is provided in a second nucleic acid construct. In other embodiments of this aspect of the invention, the sequence encoding the apolipoprotein and the sequence encoding the protein of interest are provided on the same nucleic acid construct. A DNA construct comprising a sequence encoding an apolipoprotein and a sequence encoding a protein of interest has a separate promoter for the two gene sequences and / or has an IRES sequence between the two gene sequences May be.

  In these aspects of the invention, the nucleic acid construct encoding an apolipoprotein may encode any apolipoprotein, eg, apolipoprotein AI, apolipoprotein A-II, apolipoprotein described herein. A-IV, apolipoprotein AV, apolipoprotein B-100, apolipoprotein B-48, apolipoprotein CI, apolipoprotein C-II, apolipoprotein C-III, apolipoprotein D, apolipoprotein E, apolipoprotein H, lipoprotein (a), apoliphorin I, apoliphorin II or apoliphorin III, or apolipoprotein AI, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein AV, apo Poprotein B-100, apolipoprotein B-48, apolipoprotein C-I, apolipoprotein C-II, apolipoprotein C-III, apolipoprotein D, apolipoprotein E, apolipoprotein H, lipoprotein (a), apoliphorin I , Variants of apoliphorin II or apoliphorin III, any variant of these apolipoproteins, or modified apolipoproteins having one or more domains with substantial homology to native apolipoproteins.

  The nucleic acid construct encoding an apolipoprotein may encode an apolipoprotein having an amino acid sequence obtained by modifying the amino acid sequence of a wild-type apolipoprotein. In some embodiments, the nucleic acid construct encoding an apolipoprotein or apolipoprotein variant encodes a tag sequence that is fused to the apolipoprotein sequence.

  In some preferred embodiments, the protein of interest translated using the method of the invention is a membrane protein and the amount of solubilized form of the protein of interest synthesized after incubating the in vitro protein synthesis system is the same reaction. Greater than the amount of target protein translated in the absence of apolipoprotein translation. In some preferred embodiments, the protein of interest translated using the methods of the invention is a membrane protein, and in the absence of apolipoprotein translation in the same reaction by incubating an in vitro protein synthesis system. The target soluble protein is synthesized in a higher percentage relative to the total amount of the target protein than when translated.

  In some embodiments, an in vitro protein synthesis system of the invention comprises an apolipoprotein comprising one or more nucleic acid constructs encoding a protein of interest and one or more lipids (eg, one or more phospholipids). Become. In some embodiments, the methods of the invention relate to a method of synthesizing a solubilized form of a target protein, the method comprising, in an in vitro synthesis system comprising one or more lipids, a nucleic acid construct encoding the apolipoprotein and the target protein And incubating an in vitro protein synthesis system to synthesize phospholipid-apolipoprotein particles and a target protein associated with the apolipoprotein particles. In these methods, the nucleic acid sequence encoding the apolipoprotein may be present on the same nucleic acid molecule as the sequence encoding the target protein, or encodes the apolipoprotein and target protein synthesized in an in vitro protein synthesis reaction. The sequence may be present on a separate nucleic acid molecule.

  In some embodiments of these aspects of the invention, the method further comprises isolating the protein of interest from an in vitro synthesis mixture. The isolation can be performed using, for example, an affinity reagent that binds to a tag incorporated in the sequence of the apolipoprotein or the target protein.

  In a further aspect, the present invention also relates to providing an in vitro protein synthesis system comprising a cell extract, a nucleic acid template encoding an apolipoprotein, and a nucleic acid template encoding a protein of interest. In certain embodiments, the invention includes an in vitro protein synthesis system comprising a cell extract, a first nucleic acid molecule encoding an apolipoprotein, and a second nucleic acid molecule encoding a protein of interest. . In another embodiment, the invention relates to an in vitro protein synthesis system comprising a cell extract and a nucleic acid template encoding an apolipoprotein and a protein of interest.

  In the in vitro system of the present invention, the apolipoprotein encoded by the nucleic acid template to be used may be any apolipoprotein, and the apolipoprotein used in the method of the present invention may be any apolipoprotein, for example, but not limited thereto. Apolipoprotein AI, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein AV, apolipoprotein B-100, apolipoprotein B-48, apolipoprotein CI, apolipoprotein C-II Apolipoprotein C-III, apolipoprotein D, apolipoprotein E, apolipoprotein H, lipoprotein (a), apoliphorin I, apoliphorin II or apoliphorin III, these apo All variants of lipoproteins, or modified apolipoprotein having one or more domains having substantial homology may be mentioned to the native apolipoproteins.

  The apolipoprotein sequence encoded by the nucleic acid construct used in the method and in vitro synthesis system according to the present invention may be a sequence modified from a natural or wild type sequence, and one or more deletions in the wild type apolipoprotein sequence. The sequence may be mutated or added. A construct encoding an apolipoprotein may also encode an amino acid tag that is fused in-frame with the apolipoprotein sequence. The nucleic acid template encoding an apolipoprotein may be a DNA template or an RNA template. A nucleic acid template encoding an apolipoprotein may be bound to a solid support (eg, a bead, matrix, chip, array, membrane, sheet, dish or plate).

  The nucleic acid template encoding the target protein may be a DNA template or an RNA template, and may encode any target protein, such as, but not limited to, enzymes, structural proteins, transport proteins, hormones, growth factors, inhibitors Or an activator etc. are mentioned. In some preferred embodiments, the protein of interest translated using the method of the invention is a membrane protein. The construct encoding the protein of interest may also encode an amino acid tag that fuses in-frame with the sequence of the protein of interest.

  The nucleic acid construct present in the in vitro protein synthesis system of the present invention may encode a plurality of target proteins. The nucleic acid template encoding the protein of interest may be bound to a solid support (eg, a bead, matrix, chip, array, membrane, sheet, dish or plate).

  The apolipoprotein and the protein of interest may be encoded on a single nucleic acid construct present in the in vitro synthesis system of the present invention. In these embodiments, the present invention relates to providing an in vitro protein synthesis system comprising a cell extract, an energy source, a nucleic acid template encoding an apolipoprotein, and a nucleic acid template encoding an apolipoprotein and a protein of interest.

  The in vitro protein synthesis system of the present invention may further comprise one or more chemical energy sources, free amino acids, salts, enzymes, inhibitors or cofactors. The in vitro protein synthesis system of the present invention may further comprise one or more surfactants or one or more lipids (eg, one or more phospholipids).

  The present invention also relates to providing a kit, the kit comprising a cell extract and one or more apolipoproteins or one or more nucleic acids encoding apolipoproteins. The kit further includes one or more amino acid solutions, one or more buffers, one or more salts, one or more nucleotides, one or more enzymes, one or more inhibitors, one or more energy sources, Optionally, it may comprise one or more of one or more lipids, one or more surfactants, one or more nucleic acid vectors or one or more nucleic acid constructs.

  In one embodiment of the kit of the invention, the kit is configured for in vitro protein synthesis and comprises a cell extract and one or more apolipoproteins. The apolipoprotein may be present in the cell extract or may be prepared separately as a solid or solution. Many other embodiments of the invention relate to providing one or more nucleic acid constructs encoding cell extracts and apolipoproteins. The nucleic acid construct may be an RNA construct or a DNA construct, and may be prepared as a solid (such as a lipophilic product) or a solution.

  In another embodiment of the kit of the invention, the kit is configured for in vitro protein synthesis and comprises a cell extract and one or more phospholipid-apolipoprotein particle compositions. The phospholipid-apolipoprotein particle composition may be present in the cell extract or may be prepared separately.

  The invention described herein is not limited to a particular composition or process step, but may be varied as appropriate. The section headings described herein are for convenience only and do not limit the scope of the invention.

Detailed Description of the Invention
Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates. The following terms used in this specification are defined as follows in the present invention. The singular forms “a” and “a” include plural forms of the subject matter unless the context clearly indicates otherwise. Thus, for example, the term “ligand” includes a plurality of ligands, the term “antibody” includes a plurality of antibodies, and the like.

  In the present invention, the term “about” or “approximately” in relation to any numerical value shall mean a value of ± 10% of a certain value. For example, “about 50 ° C.” (or “approximately 50 ° C.”) includes a temperature range of 45 ° C. to 55 ° C. (inclusive). Similarly, “about 100 mM” (or “approximately 100 mM”) includes a concentration range of 90 mM to 110 mM (inclusive).

  The terms “in vitro protein synthesis” (IVPS), “in vitro translation”, “cell-free translation”, “RNA template-dependent in vitro protein synthesis”, “RNA template-dependent cell-free protein synthesis” and “cell-free protein synthesis” It may be used interchangeably in the present specification, and refers to a cell-free synthesis method for all proteins. In vitro transcription-translation (IVTT) is one non-limiting example of IVPS.

  The terms “in vitro transcription” (IVT) and “cell-free transcription” may be used interchangeably herein and refer to a method of cell-free synthesis of RNA from any DNA without the synthesis of protein from RNA. And A preferred RNA is a messenger RNA (mRNA) encoding a protein.

  The terms “in vitro transcription-translation” (IVTT), “cell-free transcription-translation”, “DNA template-dependent vitro protein synthesis” and “cell-free DNA template-dependent protein synthesis” are used interchangeably herein. In other words, it refers to a method of cell-free synthesis of mRNA from any DNA (transcription) and from mRNA (translation).

  As used herein, the term “gene” refers to a nucleic acid that contains information necessary for the expression of a polypeptide, protein or untranslated RNA (eg, rRNA, tRNA, antisense RNA). When the gene encodes a protein, it comprises a promoter and an open reading frame sequence (ORF) of the structural gene and other sequences involved in protein expression. When a gene encodes RNA that is not translated, it comprises a promoter and a nucleic acid that encodes the RNA that is not translated.

  As used herein, the term “nucleic acid molecule” refers to a series of contiguous nucleotides (riboNTPs, dNTPs, ddNTPs or combinations thereof) of any length. The nucleic acid molecule may encode a full-length polypeptide or any length fragment thereof, or may be a non-coding sequence. In the present invention, the terms “nucleic acid molecule” and “polynucleotide” may be used interchangeably, and may include RNA and DNA.

  “Controllably linked” refers to a plurality of components of interest being in parallel in a relationship that allows them to function in their intended manner. For example, linking a control sequence to a coding sequence in a controllable manner refers to linking in such a manner that the coding sequence is expressed in a state compatible with the control sequence.

  As used herein, the term “polypeptide” refers to a series of contiguous amino acids of any length. The terms “peptide”, “oligopeptide” or “protein” can be used herein interchangeably with the term “polypeptide”.

  “Mutation” refers to a change that occurs in the genome of a standard wild-type sequence. Mutations include deletions, insertions or rearrangements of nucleic acid sequences at the genomic location, or may refer to single base substitutions at the genomic location, referred to as “point mutations”.

  As used herein, “substitution” refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.

  A “variant” of a polypeptide or protein used in the present invention means an amino acid sequence substituted with one or more amino acids in a subject polypeptide or protein. Preferably, a variant of a polypeptide retains one or more activities of the polypeptide. Preferably, the variant of the polypeptide has at least 60% identity to the protein of interest having at least 15 amino acid sequences. Preferably, the variant of the polypeptide has at least 70% identity to the protein of interest having at least 15 amino acid sequences. Preferably, a variant of a polypeptide has at least 80%, 90%, 95% or 99% identity to a protein of interest having at least 15 amino acid sequences. Preferably, a variant of a polypeptide has at least 80%, 90%, 95% or 99% identity to a protein of interest having at least 20 amino acid sequences. Variants may have “conservative” mutations, in which case the substituted amino acid often has the same structure or chemical properties as the pre-substituted amino acid (eg, replacement of leucine with isoleucine). . A variant may be a “non-conservative” substitution (eg, replacement of glycine with tryptophan). Similar mutations include amino acid deletions, insertions, or both. As a guideline for deciding which amino acid residue is to be substituted, inserted, or deleted without impairing biological or immunological activity, a known computer program (eg, DNASTAR software) is used. Can be detected.

  A “conservative amino acid substitution” refers to a substitution that is very unlikely to impair the properties of the original protein, i.e., the structure and particularly function of the protein is maintained and not subject to significant changes due to such substitution. Point to. In conservative amino acid substitutions, usually (a) the structure of the polypeptide backbone in the substitution region (eg β sheet or α helix structure), (b) the charged or hydrophobic nature of the molecule at the substitution site, and / or (c) the side chain The bulk (bulk) etc. is maintained. Conservative substitution includes, for example, replacement of a negatively charged amino acid with another amino acid (negatively charged amino acids include aspartic acid and glutamic acid), substitution of a positively charged amino acid with another amino acid (positive Charged amino acids include lysine and arginine), and substitution with amino acids having uncharged polar groups with similar hydrophilicity values (the first group of amino acids with similar hydrophobicity includes leucine, isoleucine And valine, the other group is glycine and alanine, the third group is asparagine and glutamine, the fourth group is serine and threonine, and the fifth group is phenylalanine and tyrosine. Group).

  “Deletion” refers to a mutation in an amino acid or nucleotide sequence that results in the loss of one or more amino acid residues or nucleotides.

  The term “derivative” refers to a polynucleotide or polypeptide that has been chemically modified. Examples of the chemical treatment of the polynucleotide include substitution of hydrogen with an alkyl group, an acyl group, a hydroxyl group, or an amino group. A derivative polynucleotide encodes a polypeptide that retains one or more biological or immunological functions of the natural molecule. Derivative polypeptides may be modified by glycosylation, PEGylation, biotinylation, or any similar method that retains one or more biological or immunological functions of the resulting polypeptide.

  As applied to a polypeptide sequence, the term “% identity” refers to the percentage of matching residues between at least two polypeptide sequence alignments using a standardized algorithm. Methods for aligning polypeptide sequences are known. Some alignment methods are based on conservative amino acid substitutions. Such conservative substitutions (described in more detail above) typically maintain the structure (ie, function) of the polypeptide because the charge and hydrophobicity at the substitution site is maintained. Percent identity may be measured over all defined polypeptide sequences, eg, as defined by a particular SEQ ID NO, or may be measured over a shorter sequence length. For example, fragment lengths taken from long sequence length defined polypeptide sequences include fragments with at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. It is understood that such lengths are exemplary and that% identity may be measured using any fragment length supported by the sequences shown herein (table, figure or sequence listing).

The% identity between polypeptide sequences can be calculated using the default parameters of the CLUSTAL V algorithm, such as those incorporated into the MEGALIGN version 3.12e sequence alignment program (described in the present invention, supra). Default parameters for pairwise sequencing of polypeptide sequences using CLUSTAL V are set as follows:
Ktuple = 1,
gap penalty = 3,
window = 5 and “diagonals saved” = 5.
The PAM250 matrix is selected as the default residue weight table. Like the polynucleotide sequence, the% identity is output by CLUSTAL V as the “percent similarity” between aligned polypeptide sequence pairs.

Alternatively, the NCBI BLAST software set may be used. For example, when performing a pair-wise comparison of two polypeptide sequences, the “BLAST 2 Sequences” tool, Version 2.0.12 (April 21, 2000) or later version Version 2.2.12 (August 2005) 28)), 2.2.13 (released on December 6, 2005), or 2.2.14 (released on May 7, 2006), with default parameters set up with blastp. Examples of such default parameters include:
Matrix: BLOSUM62,
Open Gap: 11, and Extension Gap: 1 penalties,
Gap x drop-off 50,
Expect: 10,
Word Size: 3,
Filter: on is mentioned.

  “Substantially purified” refers to a species or activity in which a species or activity is predominant (eg, more abundant on a molar basis than any other individual species or activity of the composition). Preferably, a substantially purified fraction is a composition whose composition or activity contains at least about 50% (mole, weight or activity basis) of all macromolecules or activity. Refers to that. Typically, a substantially pure composition refers to a composition that is present at about 80% or more, preferably about 85% or more, 90% or more, or 95% or more for all macromolecular species or activities.

  The terms “detectably labeled” and “labeled” may be used interchangeably herein and the molecule (eg, nucleic acid molecule, protein, nucleotide, amino acid, etc.) may be replaced with other moieties, or It is intended to indicate a state in which a molecule that generates a signal that can be detected by various detection means (for example, a measuring device, visual observation, photography, X-ray photography, etc.) is given. In such cases, various known methods such as covalent or ionic bonding, aggregation, affinity coupling (including the use of primary and / or secondary antibodies (either one or both have a detectable label)), etc. A molecule or moiety that emits a signal (“label” or “detectable label”) can be attached (or “labeled”) to the molecule. Suitable detectable labels used in the preparation of labeled or detectably labeled molecules according to the present invention include, for example, heavy isotope labels, heavy atom labels, radioisotope labels, fluorescent labels, chemiluminescence Labels, bioluminescent labels and enzyme labels and others known to those skilled in the art.

  As used herein, the term “label” refers to a chemical group or protein that can be detected directly or indirectly (eg, based on its spectral properties, form, or activity) and attached to a target or compound. Use this method. The label may be directly detectable (fluorophore) or indirectly detectable (hapten or enzyme). Such labels include, but are not limited to, identifying radioisotopes that can be measured with a radiation counter, dyes, dyes or other chromogens that can be visually observed or measured with a spectrophotometer, spin labels that can be measured with a spin label analyzer, such as , Heavy atom labels used in X-ray crystallography and NMR, eg heavy isotope labels used in mass analysis, and fluorescent labels (fluorophores) (output signals must be generated by excitation of appropriate molecular adducts) And can be visualized by absorbing light into the dye and exciting it, or it can be accurately measured, for example, with a standard fluorometer or image processing system). The label can be a chemiluminescent substance (the output signal is generated by chemical treatment of the signal compound), a metal-containing substance, or an enzyme (an enzyme-dependent secondary signal is generated (for example, a colored product from a colorless substrate). The term “label” of the present invention includes, as a special case, natural amino acids (eg, slightly fluorescent (eg, tryptophan) or UV-absorbing amino acids), although such amino acids. Is not usually encompassed by the term “label” or “detectable label.” The term “label” may also refer to a “tag” or hapten that can selectively bind to a conjugated molecule, and the conjugated molecule. Can be used to generate a detectable signal when it is added after the substrate, for example using biotin as a tag and further avidin or A conjugate of ptoavidin with horseradish peroxidase (HRP) was used to bind the tag, and a colorimetric substrate (eg, tetramethylbenzidine (TMB)) or fluorogenic substrate (eg, Amplex Red reagent, Molecular Probe) was added. The presence of HRP can be used to detect the presence of a number of labels known to those skilled in the art, as described above in “RICHARD P. HAUGLAND” Particles, fluorophores, haptens, enzymes and their colorimetric qualities, fluorescent substrates and chemiluminescent substrates, and other labels, which are described in (September 2002). But it is not limited to.

  A “tag” or “amino acid sequence tag” is a series of amino acids that can specifically bind to an affinity reagent. Examples of protein detection using tags that can be incorporated to capture proteins or affinity reagents include histidine multimers (4 or more, usually 6), FLAG tags, hemagglutinin tags, myc tags, glutathione-S- Examples include, but are not limited to, tags such as transferase, maltose binding protein, calmodulin, and chitin binding protein. Other amino acid sequence tags include tetracysteine-containing lumio tags, which can be used for protein purification or detection using tetraarsenic or diarsenic-containing reagents (US Pat. Nos. 6,054,271, 6,0083,78, 5,932,474, 64551569, WO 99/21013 (incorporated herein by reference).

  “Solid support” refers to a solid material having a surface for binding molecules, compounds, cells or other substances. The solid support may be a chip or array including a surface, for example glass, silicon, nylon, polymer, plastic, ceramic or metal. The solid support may be a membrane (eg nylon, nitrocellulose or polymer membrane) or a plate or dish, eg composed of glass, ceramic, metal or plastic, eg made of polystyrene, polypropylene, polycarbonate or polyallomer. 96 well plates. The solid support may be a bead or particle of any shape, preferably in the form of a sphere or near sphere, preferably the bead or particle is 1 mm or less, preferably 0.1-100 μm in diameter or maximum. Have a great deal. Such particles or beads can be any suitable material (eg glass or ceramic) and / or one or more polymers (eg nylon, polytetrafluoroethylene, TEFLON ™, polystyrene, polyacrylamide, sepharose, agarose, cellulose, Cellulose derivatives or dextran, etc.) and / or metals (especially paramagnetic metals such as iron).

  In the present invention, the term “associate” means to bind directly or indirectly. The first biomolecule associated with the second biomolecule is co-isolated with the second biomolecule based on the binding or mobility properties of the second biomolecule using one or more capture separation methods. can do.

  “Phospholipid-apolipoprotein particles” are a type of molecular complex, comprising one or more apolipoproteins and one or more phospholipids (phospholipids arranged in a bilayer), usually in nm units (Eg, present in a disk-like shape with a diameter of about 1 nm to about 995 nm, more typically about 2 to about 700 nm, or about 4 to about 600 nm. Natural and synthetic phospholipid-apolipoprotein particles are, for example, Pownall et al. (1978) Biochemistry 17: 1183-1188, Pownall et al. (1981) Biochemistry 20: 6630-6635, Jonas et al. (1984) J. Biol. Chem. 259: 6369-6375, Jonas et al. (1989). J. Biol.Chem.264: 4818-482 4, Jonas et al., (1993) J. Biol. Chem. 268: 156-1602, Leroy et al., (1993) J. Biol. Chem. 268: 4798-4805, Tricerri et al. (2000) Biochemistry 39: 14682-14691. Segal et al., (2002) J. Lipid Res. 43: 1688-1700, Manchekar et al., (2004) J. Biol. Chem. 279: 39757-39766, Pearson et al., (2005) J. Biol. 38576-38582.

  Other terms used in the field of recombinant nucleic acid technology, and molecular and cell biology terms used herein are generally known or obvious to those skilled in the art.

IVPS System The present invention uses known in vitro protein synthesis systems, but may comprise cell extracts of prokaryotic or eukaryotic cells. The cell extract may be derived from cells mutated with one or more genes (eg, a gene encoding a nuclease or a gene encoding a protease), or one or more endogenous or foreign genes (eg, tRNA). , Genes encoding polymerases, enzyme inhibitors, etc.) may be derived from cells that have been modified to express or overexpress. Cell extracts may be supplemented with proteins or other molecules that can prevent degradation of the template to enhance transcription or translation.

  Non-limiting examples of in vitro protein synthesis (IVPS) systems that can be used in the methods and compositions of the present invention include, but are not limited to, those described in the following literature. U.S. Pat. No. 5,478,730, Alakhov et al., "Method of preparing polypeptides in cell-free translation system", U.S. Pat. Nos. 5,665,563, 5,492,817 and 5,324,437, Becklt, et al. in eukaryotic cell-free extract ”, US Pat. No. 6,337,191, Swartz et al.,“ In Vitro Protein Synthesis using Glycolytic Intermediates as an Energy Source, US Patent No. 58, US Patent No. 58, ” v et al., title of the invention "Method of preparing polypeptides in cell-free system and device for it realization", US Pat. No. 6,670,173, Schels et al., title of the invention "Bioreaction mod 39, United States Patent No. 6670173, Schell et al. Et al., “Method for synthesis of polypeptides in cell-free systems”, US Patent Application Publication No. 2002/0168706, Chatterjee et al., Published November 14, 2002, “Improved systems in vitro is system in vitro. US Pat. No. 6,168,931, Swartz et al., Registered on Jan. 8, 2002, entitled “In vitro macromolecular biosynthetic methods using exogeneous amino acids and a novel A3. Registered on April 15th, title of invention "Enhanced in vitro synthesis of active proteins containing disbonded bonds", U.S. Patent Application Publication No. 2004/0110135, Nemetz et al., Published on June 10, 2004, med. DN "fragments for the in vitro expression of proteins", US Patent Application Publication No. 2004/0209321, Swartz et al., published on October 21, 2004, title of the invention "Methods of in vitro protein synthesis", US Patent Application No. 142/2002 , Motoda et al., Published Oct. 28, 2004, title of the invention "Method of producing template DNA and method of producing protein in cell-free protein synthesis 90, published in the United States, No. 4 , 2004 February 23, public, entitled "Method for protein expression starting from stabilized linear short DNA in cell-free in vitro transcription / translation systems with exonuclease containing lysates or in a cellular system containing exonucleases", US Patent Application Publication No. 2005/0009013 (Published January 13, 2005) and 2005/0032078 (February 10, 2005), both Rothschild et al., "Methods for the detection, analysis" is and isolation of nascent proteins ", US Patent Application Publication No. 2005/0032086, Sakyanan et al., published February 10, 2005, title of the invention" Methods of RNA and protein synthesis ", International Publication No. 00 / 5533z, Swart. Et al., Published on March 15, 2000, entitled “In vitro macromolecule biosynthesis methods using exogeneous amino acids and a novel ATP regeneration system”. All of these patents and patent applications are incorporated herein by reference.

  Preparation of cell extracts is the basis for in vitro protein synthesis from purified mRNA transcripts or from mRNA transcribed from DNA during in vitro synthesis reactions, which are also known. In order to synthesize the protein to be analyzed, the translation extract will be “programmed” with the mRNA corresponding to the gene and protein to be analyzed. mRNA may be prepared from DNA or purified form of mRNA may be added exogenously. RNA can be prepared by synthesis from cloned DNA using RNA polymerase in an in vitro reaction.

  Prokaryotic and eukaryotic cells can be used to synthesize proteins and / or nucleic acids of the invention (Pelham et al., European Journal of Biochemistry, 67: 247, 1976). Prokaryotic systems can be used to perform transcription and translation simultaneously or “coupled”. Cell extracts used for IVTT contain components necessary for transcription (producing mRNA) and translation (protein synthesis) in a single system. In such a system, the template nucleic acid molecule to be added is DNA.

  As shown in the Examples described herein, the cell-free extract used in the method of the present invention can be a prokaryotic or eukaryotic extract. Examples of eukaryotic in vitro protein synthesis (IVPS) extracts include, but are not limited to, rabbit reticulocyte lysate, wheat germ lysate, Drosophila embryo extract, scallop lysate (Storch et al., J. Comparative Physiology B, 173: 611-620, 2003), extracts from mouse brain (Campagnoni et al., J Neurochem. 28: 589-596, 1977, Gilbert et al., J Neurochem. 23: 811-818, 1974), as well as chicken brains ( Liu et al., Transactions of the Illinois State Academy of Science, Volume 68, 1975). The eukaryotic extract for IVPS may be an extract from cultured cells. The cultured cells may be of any type. As a non-limiting example, an in vitro translation system may be implemented using HeLa or CHO cell extracts.

  Eukaryotic extracts (optionally adding enzymes, substrates and / or cofactors) may be used to prepare translation products as proteins with post-translational modifications. Enzymes, substrates and / or cofactors for post-translational modifications may be added to the prokaryotic extract for IVPS. A cell-free extract may be prepared using a surfactant, and the cell or cell lysate (US 2006/0110788 (application number: 11/240, 651), the entire disclosure of which is incorporated herein by reference), or in a composition for an in vitro protein synthesis system. For example, translation extract preparations can be performed using non-ionic or zwitterionic surfactants at or slightly above the CMC concentration.

  The prokaryotic extract may be any prokaryotic cell, may be a gram negative and a gram positive bacterium, and is not limited to Escherichia (eg, E. coli), Klebsiella, Streptomyces, Streptococcus, Sigeria Staphylococcus, Erwinia, Klebsiella, Bacillus (eg B. cereus, B. subtilis and B. megaterium), Serratia, Pseudomonas (eg P. aeruginosa and P. syringae), Salmonella (eg S. C. typhi and S. tyhummurium) and Rhodobacter. Bacterial strains and serotypes suitable for the present invention include E. coli serotypes K, B, C and W. A typical prokaryotic extract is prepared from E. coli strain K-12. Cell extracts may be prepared from mutant bacterial strains, thereby making them devoid of nuclease or protease activity, or lacking the activity of one or more proteins that can interfere with the detection of purified or translated proteins (See US Patent Publication No. 2005/0136449, the entire disclosure of which is incorporated herein by reference).

  The IVPS system allows various proteins to be expressed simultaneously and rapidly in a multiplexed configuration (eg, array format) and can also be used to screen a large number of proteins. IVTT systems that use DNA templates improve the efficiency of these formats because there is no need to synthesize RNA transcripts separately and then purify them. In addition, a variety of unnatural amino acids can be used to efficiently incorporate them into specific proteins to be synthesized using the IVPS system (see, for example, Noren et al., Science 244: 182-188, 1989). reference)

  In certain embodiments, a cell extract or IVPS system using the extract is further provided by any of the components of US 2002/0168706, the entire disclosure of which is incorporated herein by reference. Comprising one or more. For example, the cell extract is one of inhibitors of one or more enzymes (eg, an enzyme selected from the group consisting of nuclease, phosphatase and polymerase) (optionally the extract is one or more enzymes (eg, Natural or wild-type extract may be modified to reduce the activity of an enzyme selected from the group consisting of nucleases, phosphatases and polymerases), and provide energy to protein and / or nucleic acid synthesis May contain at least two energy sources. In certain embodiments, the extract contains a Gam protein.

  Enzymes, substrates and / or cofactors for post-translational modifications may optionally be added to the prokaryotic or eukaryotic extract for IVPS, or may be originally present in the eukaryotic cell extract.

  IVPS usually comprises one or more amino acids in addition to the cell extract. An IVPS typically comprises a cell extract, one or more amino acids, and one or more energy sources that support translation. When the in vitro translation system is a transcription / translation system, it is preferable to further add a polymerase. When the in vitro translation system is a transcription / translation system, it is preferable to further add a polymerase. In vitro protein synthesis systems are known, including how to make and use them. In an exemplary embodiment, at least two amino acids and one or more compounds that provide energy for translation are added to the cell extract to constitute an IVPS system. In some exemplary embodiments, the IVPS comprises a cell extract, 20 natural amino acids and one or more compounds that provide energy for translation. In some preferred embodiments, the IVPS comprises at least two compounds that function as an energy source for translation (at least one may be a glycolytic intermediate). At least one amino acid provided to the IVPS system may be arbitrarily labeled, for example, one or more amino acids may be labeled with a radioisotope, and the identification amino acid may be incorporated to detect the protein as a translation product . In some embodiments, a feed solution comprising one or more additional energy sources and additional amino acids is added after the first incubation of IVPS. Feed solutions for IVPS systems and their use are described in US Patent Application Publication No. 2006/0110788, incorporated herein by reference.

  Some examples of IVPS systems and other related embodiments include the following: US Patent Application Publication No. 2002/0168706, “Improved In Vitro Synthesis Systems” (filing date: March 7, 2002). ), US Patent Application Publication No. 2005/0136449, “Compositions and Methods for Synthesizing, Purifying, and Detecting Biomolecules” (Filing Date: October 1, 2004), US Patent Application Publication No. 2006/0084136, “Product”. "Fusion Proteins by Cell-Free Protein Synthesis" (filing date: July 14, 2005) US Patent Application Publication No. 2006/0110788, “Feeding Buffers, Systems, and Methods for In Vitro Synthesis” (Filing Date: October 1, 2005), US Patent Application Publication No. 2006/0110788, “Feeding”. "Buffers, Systems, and Methods for In Vitro Synthesis" (filing date: October 1, 2005) and US Patent Application Publication No. 2006/0211083 (filing date: January 20, 2006) "Products and Processes for In Vitro Synthesis of Biomolecules "(the entire disclosure of the above patent application is incorporated herein).

  In some embodiments, the present invention uses the Invitrogen EXPRESSWAY ™ in vitro translation system (Invitrogen, Carlsbad, Calif.) And comprises a cell-free S30 extract and a translation buffer. The S30 extract contains many soluble translation components such as initiation, elongation and termination factors, ribosomes and tRNA extracted from intact cells. Translation buffers include amino acid, energy sources (eg, ATP and GTP), energy regeneration components such as phosphoenolpyruvate / pyruvate kinase, acetyl phosphate / acetate kinase or creatine phosphate / creatine kinase, and various other important cofactors. (Zubay, Ann. Rev. Genet. 7: 267-87, 1973, Pelham and Jackson, Eur J Biochem. 67: 247, 1976, and Erickson and Blobel, Methods Enzymol. 96, 38, 38). . Reaction buffer, methionine, T7 enzyme Mix and the desired DNA template (controllably bound to the T7 promoter) are mixed with the E. coli extract. After completion of transcription of the DNA template, the 5 'end of the mRNA binds to the ribosome, translation is initiated, and the encoded protein is synthesized.

Apolipoprotein The present invention relates to methods and compositions characterized by the presence of one or more apolipoproteins in an in vitro protein synthesis system. The apolipoprotein may be present in the cell extract when adding a template encoding POI, or may be added during the synthesis reaction, or the apolipoprotein is translated from the nucleic acid construct added to the IVPS system. May be.

  Apolipoprotein is a protein having a function of binding and transporting lipids in the circulatory system of animals. Sequence homology analysis between species, as well as structural analysis and prediction, indicate that apolipoproteins share a similar structure (several amphipathic helices). Thus, a variant or modified apolipoprotein provided herein typically has one, or at least 2, 3, 4 or more amphipathic helices, usually of wild type apolipoprotein. It comprises an amphipathic helix sequence, or conservative amino acid substitutions thereof. Furthermore, the mutant or modified apolipoprotein used in the methods and compositions of the present invention typically retains the ability to bind lipids.

  In the present invention, the term “apolipoprotein” is used broadly to refer to a protein that binds to lipids and is water soluble both in its free form and in the form associated with lipids. The apolipoprotein of the present invention has one or more helical domains and preferably forms or is expected to form an amphipathic helix. The apolipoprotein used in the methods and compositions of the present invention may preferably be a natural apolipoprotein (which may be a sequence variant of a natural apolipoprotein of any species, as described below). Or a modified protein having one or more helical domains having at least 90% homology with one or more helical domains of a natural apolipoprotein. The apolipoprotein used in the methods and compositions of the present invention has the property of increasing the yield of soluble membrane protein by at least 10% when present in an in vitro protein synthesis system (in vitro translation system). (Rate) is calculated as the amount of soluble protein synthesized or the percentage of soluble protein relative to the total synthetic protein.

  Apolipoproteins used in the methods and compositions of the present invention include apolipoprotein variants, having at least 10, 15, 20, 25, 50, 75, 100, 150 or 200 contiguous amino acids, any species A protein having at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% sequence identity to the wild type apolipoprotein of When present in, the solubility of one or more proteins expressed in the IVPS system is increased by at least 10%. In certain aspects, the soluble protein prepared in the IVPS system is increased by at least 15%, 20% or 25% compared to the same protein prepared in the IVPS system in the absence of apolipoprotein or a variant thereof, or Increase in a detectable manner. Apolipoprotein variants may have one or more sequence deletions or insertions relative to the native apolipoprotein. As a non-limiting example, tag sequences may be added to some apolipoprotein variants, or non-helical domains may be deleted.

  In certain embodiments, the mutant apolipoprotein is a wild-type mammalian apolipoprotein (especially apolipoprotein AI (Apo A-I), apolipoprotein A-II (Apo A-II), apolipoprotein A-IV (Apo A). -IV), apolipoprotein AV (Apo AV), apolipoprotein B-100 (Apo B-100), apolipoprotein B-48 (Apo B-48), apolipoprotein CI (Apo CI) ), Apolipoprotein C-II (Apo C-II), apolipoprotein C-III (Apo C-III), apolipoprotein D (Apo D), apolipoprotein E (Apo E), apolipoprotein H (Apo H) or Variant of lipoprotein (a) (Lp (a)) variant A.

  Some apolipoproteins (referred to as exchangeable apolipoproteins) bind reversibly to lipids and have a stable form both when bound to lipids and when not bound to lipids. Exchangeable apolipoproteins usually have a size of less than about 50 kDa and share structural similarity based on a variable number of amphipathic α-helical domains thought to bind to the surface of lipoprotein particles (Segrest et al. J. Lipid Res. 33: 141-166 (1992), Pearson et al., J. Biol. Chem. 280, 38576-38582 (2005), Boguski et al., Proc. Natl. Acad. Sci. 83: 8457-8461 (1985)). Examples of exchangeable apolipoproteins include apolipoprotein AI, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein AV, apolipoprotein CI, apolipoprotein C-II, apolipoprotein C-III, Examples include, but are not limited to, apolipoprotein E and apoliphorin III.

  The apolipoprotein used in the compositions and methods of the present invention may be of any animal origin or may be constructed based on the sequence of apolipoprotein of any animal species. In some embodiments, the apolipoprotein used in the methods of the invention is a mammalian apolipoprotein and has one or more apolipoprotein variant sequences derived from one or more mammalian apolipoprotein sequences. For example, apolipoprotein AI, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein AV, apolipoprotein B-100, apolipoprotein B-48, apolipoprotein CI, apolipoprotein C-II , Apolipoprotein C-III, apolipoprotein D, apolipoprotein E, apolipoprotein H or lipoprotein (a). The identity of these apolipoproteins used in the present invention may be derived from one or more species, and in many cases the name refers to humans as proteins. For example, the following human apolipoprotein sequences: gi37499465 (human apolipoprotein AI, SEQ ID NO: 1), human proapolipoprotein AI (SEQ ID NO: 2), human apolipoprotein A-II (gi296633, SEQ ID NO: 3) ), Human apolipoprotein A-IV (gi178759, SEQ ID NO: 4), human apolipoprotein AV (gi603913728, SEQ ID NO: 5), apolipoprotein B-100, (gi11014, SEQ ID NO: 6), apolipoprotein B-48 ( gi178732, SEQ ID NO: 7), apolipoprotein C-I (gi30583123, SEQ ID NO: 8), apolipoprotein C-II (gi37499469, SEQ ID NO: 9), apolipoprotein C-III (gi521205, SEQ ID NO: 10) Apolipoprotein D (gi54666584, SEQ ID NO: 11, gi1246096, SEQ ID NO: 12), apolipoprotein E (gi1788883, SEQ ID NO: 13), apolipoprotein H (gi178857, SEQ ID NO: 14), apolipoprotein Lp (a) (gi50331885, SEQ ID NO: 15) and variants thereof having at least 10, 15, 20, 25, 50, 75, 100, 150, or 200 contiguous amino acids, wherein at least 50, 60, 70, 75, 80, 85, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 having a sequence identity of 90, 95, 96, 97, 98 or 99%, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, Column number 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, is a apolipoprotein included in the methods and compositions of the present invention is not limited thereto.

  However, apolipoprotein AI, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein AV, apolipoprotein B-100, apolipoprotein B-48, apolipoprotein CI, apolipoprotein C-II, apolipo The designations of protein C-III, apolipoprotein D, apolipoprotein E, apolipoprotein H or lipoprotein (a) also include non-human species (mammals, fish, birds, marsupials, reptiles and amphibian species) (But not limited to) is also used herein to refer to these protein analogs. Thus, analogs of the protein referred to herein as names assigned as human proteins are similarly included as apolipoproteins of the present invention. Such apolipoproteins of the present invention and apolipoprotein variants derived from non-human species may or may not have the same name as other species.

  Non-limiting examples include any apolipoprotein A-I: eg rat (gi6978515), mouse (gi2145141), golden hamster (gi4063843), Atlantic salmon (gi64356), zebrafish (gi188858281), ducks (gi627301), puffers ( gi57157761), orangutans (gi233379768), chimpanzees (gi23337964), gorillas (gi233379766), pigs (gi47523850), baboons (gi86653), rabbits (gi71790) or mutant sequences thereof can be used. As a non-limiting example, any apolipoprotein A-Il: eg rat (gi202948), mouse (gi7304897), macaque (gi38049), bovine (gi6225059), horse (gi471115663) or mutant sequences thereof can be used. As non-limiting examples, use any apolipoprotein A-IV: eg rat (gi83392909), mouse (gi66880702), chicken (gi45384392), baboon (gi510276), pig (gi47523830), chimpanzee (gi601801) or its mutant sequence it can. As non-limiting examples, any apolipoprotein A-V: eg rat (gi18034777), mouse (gi31560003), bovine (gi766635264) or dog (gi57086253) or mutant sequences thereof can be used.

  As non-limiting examples, any apolipoprotein B: eg rat (gi61098031), chicken (gi1114013), rabbit (gi1114015), lemur (gi31555858), pig (gi951375), macaque (gi930126), squirrel (gi31555856), hallirat ( gi31555852) or mutant sequences thereof can be used.

  As a non-limiting example, any apolipoprotein CI: eg rat (gi6978521), mouse (gi6680704), macaque (gi1114017), rabbit (gi416626) or mutant sequences thereof can be used. By way of non-limiting example, any apolipoprotein C-II can be used, such as mouse (gi67553100), dog (gi50997236), macaque (gi342077), guinea pig (gi191239), bovine (gi1114019) puffer (gi74096407) or a mutant sequence thereof . As a non-limiting example, any apolipoprotein C-III: eg rat (gi8392912), mouse (gi15421856), dog (gi50997230), pig (gi506657386), bovine (gi475564119) or mutant sequences thereof can be used.

  As non-limiting examples, any apolipoprotein D: eg rat (gi287650), mouse (gi75676737), chicken (gi5869426), guinea pig (gi1110553) or deer (gi82469911) or mutant sequences thereof can be used.

  By way of non-limiting example, any apolipoprotein E: eg rat (gi20301954), mouse (gi67553102), chimpanzee (gi57113897), rhesus monkey (gi3913070), baboon (gi1766969), pig (gi3111233), cow (gi312833) or a variant thereof Arrays can be used.

  As a non-limiting example, any apolipoprotein H: for example rat (gi56971279), mouse (gi94400779), woodchuck (gi92111519), dog (gi54727921), bovine (gi27806741) or a mutant sequence thereof can be used.

  In some embodiments, the apolipoprotein used in the methods of the invention is an insect-derived apolipoprotein or has a sequence derived from the sequence of an insect apolipoprotein (eg, apoliphorin I, apoliphorin II or apoliphorin III). Such proteins may be derived from any species (e.g., Draspophila species, Manduca species, Locusta species, Lethocerus species, Ostrinia species, Bombyx species, and even other insects) Or they may be analogs of non-insect species. For example, apolipophorin (gi2498144, SEQ ID NO: 16), apolipophorin II (gi2746729, SEQ ID NO: 17), apolipophorin III (gi159948, SEQ ID NO: 18), and at least 10, 15, 20, 25, 50, 75, 100 , 150 or 200 contiguous amino acids and at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% relative to SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 Apolipoprotein variants having the following sequence identity are apolipoproteins that can be used in the compositions and methods of the present invention.

  Examples of the apolipoprotein that may be present in the IVPS system of the present invention include apolipoprotein AI, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein AV, apolipoprotein B-100, apolipoprotein B -48, apolipoprotein C-I, apolipoprotein C-II, apolipoprotein C-III, apolipoprotein D, apolipoprotein E, apolipoprotein H, lipoprotein (a), apoliphorin I, apoliphorin II or any kind of apoliphorin III Examples include, but are not limited to, analogs (including any kind of analog variants).

  In some exemplary embodiments, the apolipoprotein present in the IVPS system is a replaceable apolipoprotein, such as apolipoprotein AI, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein A-V, Apolipoprotein CI, apolipoprotein C-II, apolipoprotein C-Ill, apolipoprotein E or apoliphorin III are mentioned.

  In some embodiments, the apolipoprotein used in the compositions and methods of the present invention is apolipoprotein AI, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein AV, apolipoprotein from any species. Protein B-100, apolipoprotein B-48, apolipoprotein CI, apolipoprotein C-II, apolipoprotein C-III, apolipoprotein D, apolipoprotein E, apolipoprotein H, lipoprotein (a), apoliphorin I, It has at least 70% identity to at least 20 consecutive or adjacent amino acids of an apolipoprotein such as apoliphorin II or apoliphorin III. The apolipoprotein is, in a preferred embodiment, a continuous sequence of at least 20 amino acids, a continuous sequence of at least 30 amino acids, a continuous sequence of at least 40 amino acids, a continuous sequence of at least 50 amino acids, a continuous sequence of at least 60 amino acids, at least 70 amino acids. A sequence of at least 80 amino acids, a sequence of at least 90 amino acids, or a sequence of at least 100 amino acids has at least 70% identity to an apolipoprotein. In some preferred embodiments, the presence of an apolipoprotein in the IVPS system improves the solubility of one or more proteins synthesized in the IVPS system and is a contiguous sequence of at least 20 amino acids of the apolipoprotein, at least 30 amino acids. A continuous sequence of at least 40 amino acids, a continuous sequence of at least 50 amino acids, a continuous sequence of at least 60 amino acids, a continuous sequence of at least 70 amino acids, a continuous sequence of at least 80 amino acids, a continuous sequence of at least 90 amino acids, or at least 100 amino acids In at least 70% identity to the apolipoprotein. In some embodiments, the apolipoprotein used in the methods and compositions of the present invention is present in the IVPS system, thereby improving the solubility of one or more proteins synthesized in the IVPS system and Have at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to a contiguous sequence of at least 20 amino acids of the protein.

  In some embodiments, the apolipoprotein used in the compositions and methods of the present invention can be, for example, a replaceable apolipoprotein (eg, but not limited to any species of apolipoprotein AI, apolipoprotein A-II, apolipoprotein). Protein A-IV, apolipoprotein C-I, apolipoprotein C-II, apolipoprotein C-III, apolipoprotein E or apoliphorin III) at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids , At least 70%, at least 80%, at least 90%, at least 95% or at least 99% for a contiguous sequence of at least 70 amino acids, at least 80 amino acids or at least 100 amino acids Having the same properties. In some embodiments, the apolipoprotein used in the methods and compositions of the invention improves the solubility of one or more proteins synthesized in the IVPS system by being present in the IVPS system, and any kind of apolipoprotein Having at least 70% identity to a contiguous sequence of at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids or at least 100 amino acids.

  In some embodiments, the apolipoprotein is a mammalian apolipoprotein or a mammalian apolipoprotein (eg, but not limited to apolipoprotein AI, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein A -V, apolipoprotein B-100, apolipoprotein B-48, apolipoprotein CI, apolipoprotein C-II, apolipoprotein C-III, apolipoprotein D, apolipoprotein E, apolipoprotein H or lipoprotein (a) At least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids or at least 100 amino acids At least 70% contiguous sequence comprises at least 80%, at least 90%, at least 95% or at least 99% identity.

  In some embodiments, the apolipoprotein is an insect apolipoprotein (eg, apoliphorin I, apoliphorin II, or apoliphorin III), or at least 20 amino acids, at least 30 amino acids, at least 40 of insect apoliphorin I, apoliphorin II, or apoliphorin III. At least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to a contiguous sequence of amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids or at least 100 amino acids Have.

  In some exemplary embodiments, the apolipoprotein used in the methods and compositions of the invention has at least 90% sequence identity to at least 100 contiguous amino acids of the wild-type exchangeable apolipoprotein. A wild-type, exchangeable apolipoprotein or variant that can increase the production of soluble protein of bacterial EmrE protein or human GABA receptor protein by at least 10% in an in vitro protein synthesis reaction. In some embodiments, the apolipoprotein used in the methods and compositions of the invention is apolipoprotein AI, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein AV, apolipoprotein CI Any mutation having at least 90% sequence identity to at least 100 contiguous amino acids of apolipoprotein C-II, apolipoprotein C-III, apolipoprotein E or apoliphorin III, or wild-type exchangeable apolipoprotein And can increase the production of soluble protein of bacterial EmrE protein or human GABA receptor protein by at least 10% in an in vitro protein synthesis reaction.

  In certain specific embodiments, the apolipoprotein used in the methods and compositions of the invention has a sequence of at least 90% relative to at least 100 contiguous amino acids of apolipoprotein AI, or wild-type apolipoprotein AI. It is a variant of apolipoprotein AI with identity and can increase the production of soluble protein of bacterial EmrE protein or human GABA receptor protein by at least 10% in in vitro protein synthesis reaction.

  The apolipoproteins of the present invention also include modified apolipoproteins having at least 90% amino acid sequence identity to at least 10 residues of the helical domain of the native apolipoprotein. The invention includes modified apolipoproteins (“membrane scaffold proteins”) disclosed in US Patent Application Publication No. 2005/0182243, which is incorporated herein by reference, for example, but not limited to, MSP1 (with a histidine tag) SEQ ID NO: 19), MSP1 (SEQ ID NO: 20), MSP2 (with His tag) (SEQ ID NO: 21), MSP2 (with His tag, long linker) (SEQ ID NO: 22), MSP1D5D6 (SEQ ID NO: 23), MSP1D6D7 (SEQ ID NO: 21) 24), MAP1T4 (SEQ ID NO: 25), MSP1T5 (SEQ ID NO: 26), MSP1T6 (SEQ ID NO: 27), MSP1N1 (SEQ ID NO: 28), MSP1E3TEV (SEQ ID NO: 29), MSP1E3D1 (SEQ ID NO: 30), HisTEV-MSP2 (sequence) Number 31), MSP2N1 (allocation) No. 32), MSP2N2 (SEQ ID NO: 33), MSP2N3 (SEQ ID NO: 34), MSP2N4 (SEQ ID NO: 35), MSP2N5 (SEQ ID NO: 36), MSP2N6 (SEQ ID NO: 37), MSP2CPR (SEQ ID NO: 38), His-TEV- MSP1T2-GT (SEQ ID NO: 39), MSP1RC12 ′ (SEQ ID NO: 40), MSP1K90C (SEQ ID NO: 41), MSP1K152C (SEQ ID NO: 42) and the like.

  The apolipoprotein used in the methods and compositions of the present invention may be derived from any source, for example isolated from organs or tissues including blood, plasma or serum, may be isolated from cell culture, or It can also be expressed recombinantly before being added to an in vitro synthesis system. Preferably, the apolipoprotein is at least partially purified before being added to the in vitro synthesis system.

  The amino acid sequence of the apolipoprotein used in the method and composition of the present invention was modified by wild-type apolipoprotein sequence by deleting or adding one or more amino acids to the wild-type sequence, or by amino acid substitution. So that when the apolipoprotein is present in an in vitro protein synthesis reaction, it has the property of increasing the yield of a solubilized form of the protein prepared in the in vitro protein synthesis reaction. .

  For example, the apolipoprotein used in the method or composition of the invention may be deleted at the N-terminus or C-terminus, or have one or more deletions or insertions within the wild-type apolipoprotein sequence. Also good. The apolipoprotein used in the methods and compositions of the present invention may be a multimer of apolipoprotein or part thereof, for example, two or more copies of apolipoprotein joined by a linker, or a variant or part thereof. There may be. The apolipoprotein used in the methods and compositions of the present invention may be a chimeric apolipoprotein or may comprise sequences of two different apolipoproteins (or variants thereof). Furthermore, the apolipoprotein may be linked to peptides or other protein sequences as part of the fusion protein. For example, the peptide sequence may be a purification and / or detection tag.

  In some embodiments of the invention, the apolipoprotein used in IVPS is US Pat. No. 7,048,949 A1, US Patent Application Publication No. 2005/0182243 A1, 2005/0152984 A1, 2004/0053384 A1, and A membrane scaffold protein (MSP) based on the sequence of apolipoprotein A-1 disclosed in 2006/0088524 A1 (the entire disclosure of which is incorporated herein by reference).

  The apolipoprotein provided herein may bind to lipids or may be free apolipoprotein that does not bind lipids. For example, apolipoprotein may be isolated from an organ (eg, blood or plasma), from cultured cells or media, or from bacterial cells that have been modified to express recombinant apolipoprotein. Isolated apolipoproteins can be conjugated to lipids using known techniques (Pownall et al., (1978) Biochemistry 17: 1181-1888, Pownall et al. (1981) Biochemistry 20: 6630-6635, Jonas. (1984) J. Biol. Chem. 259: 6369-6375, Jonas et al., (1989) J. Biol. Chem. 264: 4818-4824, Jonas et al., (1993) J. Biol. -1602, Tricerri et al., (2000) Biochemistry 39: 14682-14691, Segall et al., (2002) J. Lipid Res. 43: 1688-1700, Pearson et al., (2 05) J.Biol.Chem.280: 38576-38582, which is incorporated herein for reference the entire disclosure).. In some embodiments of the invention, the apolipoprotein may be provided to the in vitro protein synthesis system in a manner further comprising one or more lipids (eg, one or more phospholipids). Cholesterol, cholesterol esters or one or more other neutral lipids (such as but not limited to sterol esters, mono-, di- or triacylglycerides, or acylglycerols) may optionally be added. The lipid may be present at a concentration of about 1 μg / mL to about 20 mg / mL, or about 5 μg / mL to about 10 mg / mL, or about 10 μg / mL to about 5 mg / mL. One or more phospholipids may bind to the apolipoprotein in an in vitro protein synthesis system. In some embodiments of the invention, apolipoproteins are translated using an in vitro protein system that includes one or more lipids (eg, one or more phospholipids). Apolipoproteins synthesized in cell-free systems can bind to one or more lipids during or after translation.

Phospholipid-apolipoprotein particles In some embodiments of the invention, the apolipoprotein may be present in the in vitro protein synthesis system as a phospholipid-apolipoprotein particle, in particular, the particle comprises a phospholipid. And configured as a double layer disc with apolipoprotein. Several examples of phospholipid-apolipoprotein particles, as well as methods for preparing phospholipid-apolipoprotein discs (including phospholipid apolipoprotein discs including apolipoprotein variants) are known in the prior art, eg, Jonas (1984) J. et al. Biol. Chem. 259: 6369-6375, Jonas et al. (1989) J. MoI. Biol. Chem. 264: 4818-4824, Jonas et al. (1993) J. MoI. Biol. Chem. 268: 1596-1602, U.S. Pat. No. 7,048,949, U.S. Patent Application Publication Nos. 2005/0182243 A1, 2005/0152984 A1, 2004/0053384 A1, and 2006/0088524 A1 (all disclosed herein). The contents of which are incorporated by reference).

  Nanoscale bilayer discs disclosed herein as phospholipid-apolipoprotein particles or PAP are described in US Pat. No. 7,048,949, US Patent Application Publication Nos. 2005/0182243, 2005/0152984, 2004 /. No. 0053384 and WO 02/040501 (the entire disclosure of which is incorporated herein by reference), with particular reference to nanoscale phospholipid bilayer discs, components thereof, methods of making and using the same. Yes. In the method of the invention, membrane proteins are produced that are inserted into phospholipid-apolipoprotein particles or nanoscale phospholipid bilayer discs. Add nucleic acid template to in vitro protein synthesis system including cell extract and PAP preparation and incubate in vitro protein synthesis system to synthesize solubilized form of membrane protein (solubilized form of membrane protein is inserted into PAP) To do.

  The present invention relates to the provision of translation systems and methods comprising phospholipid bilayer particles or disks containing apolipoproteins. Preferably, the apolipoprotein provided as a phospholipid-apolipoprotein has one or more amphipathic helical domains. Examples of the apolipoprotein include apolipoprotein AI, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein AV, apolipoprotein B-100, apolipoprotein B-48, apolipoprotein CI, apolipoprotein C -II, apolipoprotein C-III, apolipoprotein D, apolipoprotein E, apolipoprotein H, lipoprotein (a), apolipophorin I (apolipophorin II) or apolipophorin III, or a derivative or variant thereof (eg, chimeric apolipoporin) Protein, apolipoprotein with C-terminal or N-terminal deletion, apolipoprotein with internal deletion, apolipoprotein containing further amino acid sequence or mutated amino acid sequence) . In a preferred embodiment, the IVPS phospholipid-apolipoprotein particles of the invention comprise ApoA-I, ApoA-IV, ApoA-V, ApoC-I, ApoC-II, ApoC-III, Apo-E or apolipophorin III or Any of these variants. In some embodiments, the length of the amphipathic helical domain of the apolipoprotein can be adjusted accordingly to activate the formation of phospholipid-apolipoprotein particles having various diameters. This is considered to be advantageous when appropriately incorporating various types of proteins into the phospholipid-apolipoprotein particles.

  Phospholipids used to form phospholipid-apolipoprotein particles or discs in translation systems may be glycerol or sphingolipid-based, such as two molecules of saturated fatty acids of 6-20 carbon atoms, and common It may comprise a head group used in (for example, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine). The head group may be uncharged, positively charged, negatively charged, or zwitterion. The phospholipid may be a natural product (naturally occurring) or a synthetic product (not naturally occurring), or a mixture of a natural product and a synthetic product. Non-limiting examples of phospholipids include PC (phosphatidylcholine), PE (phosphatidylethanolamine), PI (phosphatidylinositol), DPPC (dipalmitoyl-phosphatidylcholine), DMPC (dimyristoylphosphatidylcholine), POPC (1-palmitoyl- 2-oleoyl-phosphatidylcholine), DHPC (dihexanoylphosphatidylcholine), dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylinositol, dimyristoylphosphatidylethanolamine, dimyristoylphosphatidylinositol, dihexanoylphosphatidylethanolamine, dihexanoylphosphatidylinoamine 1-palmitoyl-2-oleoyl-phosphatidyl ester Noruamin, or 1-palmitoyl-2-oleoyl - but phosphatidyl group inositol include, but are not limited to.

  As described in the prior art, isolated apolipoproteins and phospholipids may be mixed and aggregated as phospholipid-apolipoproteins, the method of which is incorporated herein by reference, Jonas et al., (1984) J. Org. Biol. Chem. 259: 6369-6375, Jonas et al. (1989) J. MoI. Biol. Chem. 264: 4818-4824, Jonas et al. (1993) J. MoI. Biol. Chem. 268: 1596-1602, U.S. Patent No. 7048949, U.S. Patent Application Publication No. 2005/0182243 A1, 2005/0152984 A1, 2004/0053384 A1, and 2006/0088524 A1. In addition, phospholipid-apolipoprotein particles are added into the cell extract or IVPS system.

  In some other inventive aspects, a nucleic acid construct encoding an apolipoprotein is added into an IVPS system comprising one or more phospholipids, the in vitro translated apolipoprotein associates with the phospholipid, and the IVPS Form phospholipid-apolipoprotein powder particles in the system.

Recombinant Cloning A cloning system that utilizes recombination at certain recombination sites, including the GATEWAY® recombinant cloning system, vectors, enzymes, and kits, commercially available from Invitrogen (Carlsbad, Calif.), 1998. U.S. Patent Application No. 09/177387 filed on October 23, 2000, U.S. Patent Application No. 09/517466 filed on March 2, 2000, and U.S. Pat. Nos. 5,888,732 and 6,277,608 (in this application). The entire disclosure is incorporated by reference). Using these systems, the MPOI coding sequence and / or the apolipoprotein coding sequence is cloned into an expression vector for in vitro translation and two or more in a single reaction using a multi-site GATEWAY® vector. A multi-open reading frame can be provided for simultaneous translation of proteins.

  Briefly, the GATEWAY® cloning system utilizes a vector that includes one or more recombination sites for cloning the desired nucleic acid molecule in vivo or in vitro. More specifically, a vector is used that contains at least two different site-specific recombination sites (eg, att1 and att2) based on the bacteriophage λ system, mutated in the wild-type (att0) site. Each mutation site has a unique property (ie its binding partner recombination site) derived from the same type of partner as the att site (eg attB1 for attP1 or attL1 for attR1) The cross-reaction with the mutant type recombination site or wild-type att0 site does not occur. Each site specificity allows directional cloning or binding of the desired molecule, thereby providing the desired orientation for the cloned molecule. Cloning and subcloning nucleic acid fragments with recombination sites at both ends using the GATEWAY system to replace selectable markers (eg ccdB) with att sites on the recipient plasmid molecule called Destination vector . Further, a desired clone is selected by transformation of a ccdB sensitive host strain and positive selection using a marker on the receptor molecule. A similar strategy can be used for negative selection with other organisms (eg, the use of toxic genes), for example, thymidine kinase (TK) in mammalian and insect cells.

In vitro protein synthesis method and system using apolipoprotein The present invention relates to a method for translating a membrane protein in the presence of a phospholipid-apolipoprotein particle (PAP). It is based on the discovery that it is inserted into a phospholipid bilayer disc). As illustrated in the examples provided herein, in an in vitro protein synthesis (IVPS) system comprising PAP, the synthesis of the membrane protein of interest (MPOI) results in the incorporation of MPOI into PAP resulting in solubility. Resulting in the production of improved MPOI.

  The inventor can also translate a membrane protein in the presence of an apolipoprotein that is not part of PAP, and the MPOI translated in the presence of the apolipoprotein is more than the same MPOI translated in vitro in the absence of the apolipoprotein. It was found that the solubility was improved. That is, the present invention relates to the provision of an in vitro synthesis method and system for translating a protein in the presence of an apolipoprotein. The present invention relates to an in vitro synthesis method and system for translating proteins in the presence of apolipoprotein, wherein the apolipoprotein in the IVPS system is not provided to PAP. The invention also relates to in vitro synthesis methods and systems for translating proteins in the presence of apolipoproteins, wherein exogenous phospholipids are not present in the IVPS system.

  Yet another feature of the present invention is that when the apolipoprotein is expressed in the same IVPS system where the MPOI is translated and the MPOI and the apolipoprotein are synthesized in the same IVPS reaction, the apolipoprotein does not contain or is apolipoprotein It is based on the discovery that the solubility of MPOI can be improved over when synthesized in a template-free IVPS reaction.

  That is, in one aspect, the present invention relates to the provision of a method for synthesizing a target protein in vitro, which includes adding a nucleic acid template encoding the target protein to an in vitro protein synthesis system containing an apolipoprotein and incubating the in vitro protein synthesis system. To synthesize a target protein. In some preferred embodiments, at least a portion of the protein of interest is synthesized in solubilized form.

  For example, the protein of interest ("POI") expressed in the IVPS system may be any protein of interest, enzyme, structural protein, transport protein, binding protein, antibody, hormone, growth factor, receptor, inhibitor or activator Etc. The examples provided herein show that the presence of apolipoprotein in the IVPS reaction does not deleteriously affect the translation of non-membrane proteins. In some preferred embodiments, the protein of interest translated using the methods of the invention is a membrane protein (“MPOI”), or a protein whose native state is associated with a biological membrane (eg, transmembrane protein, integral membrane) Type protein or membrane surface protein).

  In some preferred embodiments, the protein of interest translated using the methods of the invention is a membrane protein and the membrane of interest protein (MPOI) synthesized after incubating the in vitro protein synthesis system is free of apolipoproteins. It is obtained in more solubilized form than the protein that is translated in some cases. In a preferred embodiment, MPOI is present in the presence of apolipoprotein, such as at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, in the absence of apolipoprotein in the IVPS reaction. Synthesized in a manner that is at least 70%, at least 80%, at least 90% or at least 100% more. In some preferred embodiments, after incubating the IVPS system, the amount of soluble MPOI relative to the total protein of interest is synthesized at a higher percentage than if MPOI was translated in the absence of apolipoprotein. In a preferred embodiment, the percentage of soluble MPOI relative to total MPOI when synthesizing MPOI in the IVPS reaction in the presence of apolipoprotein is the total MPOI when synthesizing MPOI in the IVPS reaction in the absence of apolipoprotein. For example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% greater than the percentage of soluble MPOI relative to .

  As described herein, an apolipoprotein provided in an IVPS system may be a natural apolipoprotein (which may be of any species origin), a mutant sequence of a natural apolipoprotein, or a helical form of a natural apolipoprotein. It may be any modified protein having one or more helical domains with at least 90% homology to the domain. The apolipoprotein used in the methods and compositions of the present invention has the property of increasing the soluble yield of membrane protein by at least 10%. The soluble yield is the amount of soluble protein synthesized or soluble relative to the total protein synthesized when the apolipoprotein is provided to the IVPS system or is translated simultaneously in the IVPS where the membrane protein is also translated. Calculated as a percentage of protein.

  The apolipoprotein present in the IVPS system may be present in any concentration that allows translation of MPOI. Apolipoproteins that can be provided to the IVPS system, but are about 0.5 μg / mL to about 2 mg / mL, or about 1 μg / mL to about 1 mg / mL, or about 5 μg / mL to about 500 μg / mL, or Concentration from about 10 μg / mL to about 250 μg / mL. Multiple apolipoproteins may be present in a single IVPS reaction.

  One or more apolipoproteins may be added to the IVPS reaction after the nucleic acid template is added to the reaction, but are preferably present in the IVPS reaction when the nucleic acid template encoding POI is added. In the present invention, the term “add system to IVPS” refers to adding to a cell extract prepared for IVPS, even if other ingredients for in vitro synthesis have already been added or are still added. It does not have to be.

  Thus, the present invention, in another aspect, relates to the provision of a cell extract for in vitro translation comprising one or more apolipoproteins as described herein. Cell extracts for in vitro translation include all those described herein. In some embodiments, the present invention includes an IVPS system comprising an apolipoprotein, a cell extract and a chemical energy source. In some embodiments, the present invention includes an IVPS system comprising an apolipoprotein, a cell extract, a chemical energy source, and one or more amino acids. In some embodiments, the present invention includes an IVPS system comprising an apolipoprotein, a cell extract, a chemical energy source, one or more amino acids, and a nucleic acid template. The IVPS system may optionally comprise one or more lipids, surfactants, salts, buffer compounds, enzymes, inhibitors or cofactors.

  In some embodiments of the methods of the invention, the apolipoprotein is added to an IVPS system that includes one or more lipids (eg, one or more phospholipids). In some embodiments of the methods of the invention, the apolipoprotein is added to an IVPS system that includes one or more lipids, and the apolipoprotein forms an association with one or more lipids of the IVPS system. In some embodiments of the methods of the invention, apolipoprotein is associated with one or more lipids when added to the IVPS system. In some embodiments of the invention, the apolipoprotein is added to an IVPS system that includes one or more lipids, or the apolipoprotein associates with one or more lipids when added to the IVPS system, and the IVPS During the incubation of the system, the synthesized protein of interest forms an association with the apolipoprotein and its associated one or more lipids in the IVPS system.

  In some embodiments of the methods of the invention, the apolipoprotein added to the IVPS system is added as phospholipid-apolipoprotein particles (PAP). In some embodiments of the methods of the invention, the apolipoprotein added to the IVPS system is added as PAP, and the MPOI synthesized in the system forms an association with PAP.

  Thus, a further aspect of the present invention includes a cell extract for translation containing phospholipid-apolipoprotein particles (PAP) as described herein. Cell extracts for in vitro translation include all those described herein. In some embodiments, the present invention includes an IVPS system that includes a PAP, a cell extract, and a chemical energy source. In some embodiments, the invention includes an IVPS system that includes PAP, a cell extract, a chemical energy source, and one or more amino acids. In some embodiments, the invention includes an IVPS system that includes PAP, a cell extract, a chemical energy source, one or more amino acids, and a nucleic acid template. The IVPS system may optionally comprise one or more lipids, surfactants, salts, buffer compounds, enzymes, inhibitors or cofactors.

  The phospholipid-apolipoprotein particles (PAP) described in detail above may be added to the IVPS system at any concentration that allows in vitro translation, but at a concentration that enhances the solubility of MPOI translated by IVPS. It is preferable to do this. Although it is a guideline until tired, PAP is about 0.5 μg / mL to about 2 mg / mL, about 1 μg / mL to about 1 mg / mL, about 5 μg / mL to about 500 μg / mL, or about 10 μg / mL to about 250 μg / mL. You may add suitably by the density | concentration of mL (the said density | concentration is a density | concentration with respect to protein content of PAP). Multiple types of PAP may be present in a single IVPS reaction, ie different PAPs may contain different apolipoproteins and / or different phospholipid compositions.

  The present invention relates to the provision of efficient systems and methods for synthesizing solubilized forms of cell-free system membrane proteins. The method comprises expressing the membrane protein in a cell free system comprising phospholipid-apolipoprotein particles.

  In some embodiments of the invention, the method further comprises isolating the protein of interest from the IVPS mixture. For example, the isolation method may use a peptide tag as a part of the apolipoprotein, or may use a peptide tag as a part of the target protein.

  Apolipoprotein can be provided to the IVPS system by translating it with an IVPS system that translates POI. Therefore, in yet another aspect, the present invention relates to providing a method for synthesizing a protein in vitro, the method comprising adding a nucleic acid construct encoding an apolipoprotein and a nucleic acid construct encoding a protein of interest to an in vitro synthesis system, Incubating the in vitro protein synthesis system and synthesizing the apolipoprotein and the protein of interest. In some preferred embodiments, the protein of interest is synthesized in solubilized form. In some preferred embodiments, the protein of interest is a membrane protein.

  In some embodiments, the apolipoprotein is provided by a first nucleic acid construct and the protein of interest is provided by a second nucleic acid construct. In other embodiments of this aspect of the invention, the sequence encoding the apolipoprotein and the sequence encoding the protein of interest are provided on the same nucleic acid construct. Optionally, a GATEWAY® vector and a cloning system can be used to create a nucleic acid construct encoding one or both of the desired apolipoprotein and protein. In some embodiments, a DNA construct comprising a sequence encoding an apolipoprotein and a sequence encoding a protein of interest comprises a first promoter for the coding sequence of the apolipoprotein and a second for the coding sequence of the protein of interest. Has a promoter. In one variation, a nucleic acid construct comprising a sequence encoding an apolipoprotein and a sequence encoding a protein of interest has an IRES sequence between the two coding sequences.

  In these aspects of the invention, the nucleic acid construct encoding the apolipoprotein is disclosed herein as any apolipoprotein (natural apolipoprotein, natural apolipoprotein mutant sequence, or a helical domain of a natural apolipoprotein). Including modified apolipoproteins having one or more helical domains having at least 90% homology to them). The nucleic acid construct encoding the apolipoprotein may encode an apolipoprotein having an amino acid sequence obtained by modifying the amino acid sequence of the wild-type apolipoprotein. In some embodiments, the nucleic acid construct encoding an apolipoprotein or apolipoprotein variant encodes a tag sequence that is fused to the apolipoprotein sequence.

  In some preferred embodiments, the target protein is translated in an IVPS comprising a template encoding an apolipoprotein and a template encoding a membrane protein, and no apolipoprotein is present or produced in the same reaction after incubating the in vitro protein synthesis system. Solubilized forms of MPOI are synthesized in greater amounts than if the desired membrane protein (MPOI) is translated under non-allowed conditions. In some preferred embodiments, the protein of interest translated using the method of the invention is a membrane protein, and after incubation of the IVPS system, the protein of interest under conditions in which no apolipoprotein is present or produced in the same reaction In a higher percentage than is translated, the solubilized form of the protein of interest is synthesized.

  In some embodiments, an in vitro protein synthesis system of the invention comprising one or more nucleic acid constructs encoding a protein of interest and an apolipoprotein comprises one or more lipids (eg, one or more phospholipids). . In some embodiments, a method of the invention comprising synthesizing a protein of interest in a solubilized form comprises adding a nucleic acid construct encoding the apolipoprotein and the protein of interest to an in vitro synthesis system comprising one or more lipids. Adding the encoding nucleic acid construct, incubating the in vitro protein synthesis system, and synthesizing the target protein associated with the apolipoprotein particles and the phospholipid-apolipoprotein particles.

  A further aspect of the present invention relates to the provision of an in vitro protein synthesis system comprising a cell extract, a nucleic acid template encoding an apolipoprotein and a nucleic acid template encoding a protein of interest. In certain embodiments, the invention includes an in vitro protein synthesis system comprising a cell extract, a first nucleic acid molecule encoding an apolipoprotein, and a second nucleic acid molecule encoding a protein of interest. In other embodiments, an in vitro protein synthesis system comprising a cell extract and a nucleic acid template encoding an apolipoprotein and a protein of interest is included. The nucleic acid template may be either DNA or RNA or both.

  The apolipoprotein sequence encoded by the nucleic acid construct used in the methods and in vitro synthesis systems of the present invention can be any apolipoprotein sequence disclosed herein. A construct encoding an apolipoprotein may encode an amino acid tag that fuses in-frame with the apolipoprotein sequence. The nucleic acid template encoding apolipoprotein may be a DNA template or an RNA template. For example, a nucleic acid template encoding an apolipoprotein may be bound to a solid support (bead, matrix, chip, array, membrane, sheet, dish or plate).

  The nucleic acid template encoding the target protein may be a DNA template or an RNA template, and may encode any target protein (eg, enzyme, structural protein, transport protein, hormone, growth factor, inhibitor or activator) . In some preferred embodiments, the protein of interest translated using the method of the invention is a membrane protein. The construct encoding the protein of interest may encode an amino acid tag that is fused in frame with the protein sequence of interest.

  The nucleic acid construct present in the in vitro protein synthesis system of the present invention can encode a plurality of proteins of interest. For example, a nucleic acid template encoding a protein of interest may be bound to a solid support (beads, matrix, chip, array, membrane, sheet, dish or plate).

  The present invention also relates to methods for efficient systems and methods for synthesizing soluble and easily purifiable forms of membrane proteins in vitro. In these methods, MPOI is synthesized in an in vitro translation reaction involving apolipoprotein, and the apolipoprotein has a purification tag. Capturing apolipoprotein using a purification tag allows co-isolation with membrane proteins synthesized in vitro in the presence of apolipoprotein. In embodiments where apolipoprotein is incorporated into PAP, capture of the apolipoprotein using a purification tag allows isolation of PAP containing MPOI. PAP incorporating MPOI can be used for many assays, as well as for structural analysis (eg, NMR or X-ray crystallography).

  In other embodiments, the (MPOI) membrane protein of interest can optionally be translated in the presence of apolipoproteins, so that the MPOI can further identify, isolate, bind or purify, or immobilize the synthesized protein. A protein tag may be bound to the. In this case, the apolipoprotein may optionally have a tag.

  The invention further relates to the provision of an in vitro method for the synthesis of POI (including MPOI) (protein identity may or may not be known) in an IVPS reaction involving apolipoprotein (around PAP or not around PAP). There, many reactions are performed in parallel (eg, in multi-well plates), resulting in many soluble proteins for the assay. The protein may be expressed from a template on a vector, and the vector has a sequence for expression by transcription and translation in the vicinity of the cloning site. A vector may be used to clone a library of sequences, optionally comprising a protein tag sequence that can be expressed in-frame with the POI.

  In a preferred embodiment, the apolipoprotein of PAP is an affinity tag (eg, His tag, glutathione) used to bind PAP containing MPOI to a solid support (eg, microwell surface, chip surface, sheet, membrane, matrix or bead). Tag, streptavidin tag, etc.). MPOIs translated with PAP may be immobilized on microwells, chip surfaces, sheets, membranes, matrices or beads via their insertion into the binding PAP. Before or after translation of the MPOI, PAP may be bound to a solid support in the presence of PAP.

  That is, the method of the present invention may be used to make membrane protein arrays or multiwell assay plates, where individual MPOIs inserted at specific sites on the array by localized in vitro translation reactions involving PAP. It is possible to bind PAP having Such arrays can be used to perform many types of screening and assays, including but not limited to enzyme assays, ion channel assays, and drug binding tests. Labeling of MPOI in a translation reaction as described below may be performed to simplify the array assay.

  Array or multiwell assay plates may be prepared by in vitro translation reactions performed on the array or plate. For example, an IVPS reaction solution containing a nucleic acid template encoding cell extract, PAP and MPOI may be added to each site on the array (or well or plate). PAP may be attached to the array via His, glutathione, streptavidin or other tags modified to the apolipoprotein. The MPOI may be a known or unknown protein.

  In another embodiment, the MPOI may be cloned into a vector that provides a sequence encoding a tag as the N-terminal or C-terminal amino acid sequence of the protein of interest. Proteins that can be translated in the presence of apolipoproteins that can be prepared without associated phospholipids or in the context of PAP may be further isolated, bound or purified, or immobilized using the tag. The synthesized protein can be captured, for example, on the bottom of a well treated or coated with an affinity capture reagent, or a location or well in an array, or a filter, matrix, or bead.

  The present invention also includes a method for translating membrane proteins in an IVPS system that includes apolipoproteins, and in particular, MPOI can be identified as a radioisotope, heavy isotope label or fluorescent label (eg, BODIPY (registered on an amino group)). (Trademark) characterized by labeling during translation, such as by using tRNA methionine (fmet) with FL fluorophore (which is misaminoacylated) to introduce BODIPY® FL fluorophore at the N-terminus), etc. To do. Alternatively, the MPOI may be modified in a manner that includes a tag that can bind to a label (eg, a fluorescent label) (as a non-limiting example, the LUMIO ™ tetracysteine sequence motif detection technology (the entire disclosure herein is Incorporated Invitrogen, Carlsbad, CA, for example, US Patent Application Publication No. 2003/0083373, US Pat. No. 5,932,474, US Pat. No. 6,0083,782, US Pat. No. 6,054,271, International Publication No. PRO-Q® Sapphire 532, 365 or 488 horigohistidine staining for Carlsbad, CA) can be used.) The method can be incorporated into apolipoproteins and synthesized membrane proteins. In an alternative embodiment, the method comprises translating the membrane protein in an in vitro synthesis reaction comprising one or more apolipoproteins. In particular, the translated membrane protein has one or more tags that can bind to the label, and the method allows production of a solubilized form of the labeled or tagged membrane protein. The method in the embodiment allows for the production of tagged and / or labeled membrane proteins with improved solubility.

  In some preferred embodiments of these methods, the apolipoprotein present in the IVPS system is present in PAP. That is, the present invention translates a membrane protein in an in vitro synthesis reaction that includes one or more labels that can be incorporated into the phospholipid-apolipoprotein particles and the synthesized membrane protein to prepare the labeled membrane protein. Is included. The method translates the membrane protein in an in vitro synthesis reaction that includes one or more labels that can be incorporated into the phospholipid-apolipoprotein particle and the synthesized membrane protein, and is inserted into the phospholipid-apolipoprotein particle. Preparing a labeled membrane protein. In an alternative embodiment, the method comprises translating the membrane protein in an in vitro synthesis reaction comprising one or more phospholipid-apolipoprotein particles, in particular, labeling within the translated membrane protein. Including one or more tags that can be combined with. The method comprises translating a membrane protein in an in vitro synthesis reaction that includes phospholipid-apolipoprotein particles, and in particular, includes one or more tags that can bind to a label within the translated membrane protein. A membrane protein with a tag in a state of being inserted into a phospholipid-apolipoprotein particle is obtained.

  The labeling of the membrane protein inserted into the PAP makes it possible to analyze the membrane protein-ligand binding, and in particular affects the fluorescence properties of the labeled protein due to the ligand binding. In related embodiments, ligand-membrane protein binding can be assessed by labeling the ligand and using a fluorescence detection method such as FRET. That is, the present invention includes a method of translating a membrane protein with an IVPS system containing PAP, wherein a label or tag that can be directly or indirectly bound to the label is incorporated into the translated membrane protein.

  Consideration of membrane protein-protein interactions when labeling the membrane protein inserted into the PAP, including but not limited to, where the protein-protein interaction affects the fluorescent properties of the labeled protein (eg, receptor Any protein-protein interaction such as dimerization) can be studied. One or both of the proteins may be labeled.

  Assays (including but not limited to assays for ligand binding, ion channel activity and protein-protein interactions) may be performed on the array, which includes PAP with an MPOI inserted. In this case, since the assay on the membrane protein can be performed at a high throughput, a troublesome and routine purification step can be omitted.

  The present invention also includes a method of incorporating two or more different membrane proteins of interest into normal PAP using in vitro translation methods. In these embodiments, different membrane proteins can be expressed in normal in vitro reactions using the same or different nucleic acid template molecules. For example, a multi-site GATEWAY® vector (Invitrogen, Carlsbad, Calif.) Can be used to clone in at least two open reading frames on the same vector. During translation, a label may be incorporated into the protein, or separate proteins with separate tags may be synthesized and used for conjugation with separate labeling reagents. Thus, using fluorescence measurements such as, but not limited to, FRET and TRET, protein-protein interactions of phospholipid bilayers, particularly protein-protein interactions that occur within protein complexes with many proteins. You may monitor. In some aspects of the present invention, the IVPS system may include a cell extract and a nanoscale phospholipid bilayer disc, specifically the nanoscale phospholipid bilayer disc comprising components involved in protein translocation mechanisms. Is included. Constituents involved in the protein transfer mechanism include the Sec YEG protein, and also include mammalian counterparts, protein transfer (pore formation) proteins, SRP receptors, ribosome receptors, etc. Promoted. It is considered that another protein (SecA, SecB or FtsY (in it)) may be externally added to the reaction system. Chaperonins may be added to promote protein folding and membrane insertion.

  Membrane protein components that constitute the protein transfer mechanism may be provided to a pre-prepared PAP, in which case the protein involved in protein transfer is inserted by a solubilization / dialysis method to prepare the PAP. Or may be inserted into the PAP using an in vitro translation system, as described herein.

  The present invention also encompasses IVPS systems and methods that include a PAP component (ie, a phospholipid and a solubilized form of an apolipoprotein), in particular, MPOI is translated in the presence of the PAP component, and PAP is associated with MPOI. The reaction associates, resulting in PAP with incorporated MPOI. A method for producing PAP or “nanodisc” is disclosed in, for example, US Patent Application Publication No. 2005/0182243. The present invention provides a solubilized PAP component (such as but not limited to apolipoprotein, disclosed herein and disclosed in US Patent Application Publication No. 2005/0182243) and a phospholipid during an IVPS reaction, and a nucleic acid template encoding MPOI Providing the MPOI translated in the presence of the PAP component and incorporated into the PAP during the translation reaction. Aggregation of PAP can occur before the translation reaction of MPOI, during translation, or after translation.

  The method of preparing PAP by providing components to the IVPS system can be combined with other embodiments described herein, such as the use of tagged apolipoproteins, on arrays or on multiwell plates Translation of MPOI using a PAP component, translation of two or more MPOIs using a PAP component, addition of a component related to protein transfer to an IVPS reaction mixture containing the PAP or PAP component, and PAP or PAP component Combinations with translation of one or more protein transfer related components in an IVPS reaction mixture comprising.

Apolipoprotein-membrane protein-containing composition Another embodiment of the present invention relates to the provision of a composition comprising one or more membrane proteins associated with one or more apolipoproteins. Typically, the composition is a solubilized and isolated complex in aqueous solution with one or more apolipoproteins and one or more membrane proteins. The complex may comprise a lipid (eg, phospholipid). The complex of membrane protein and apolipoprotein may be substantially lipid free in some embodiments. Complex membrane proteins are typically synthesized using an in vitro protein synthesis system in the presence of apolipoprotein as disclosed herein. Surfactant-free ones can be mentioned as a complex that exemplifies this embodiment of the present invention. The complex comprises an apolipoprotein, a full length or part of a membrane protein (usually including at least the N-terminal portion), one or more ribosomes, and one or more RNA molecules (eg, RNA molecules encoding membrane proteins). A cell-free complex may be included. The complex may contain lipid or be substantially lipid free. The complex may be an isolated complex. The complex may be bound to the solid support via a nucleic acid template encoding apolipoprotein or membrane protein, or apolipoprotein or membrane protein, either of which may optionally comprise a peptide tag You may combine through.

  The following examples illustrate exemplary embodiments of the present invention but do not limit the invention in any way.

Example 1 In Vitro Expression of Non-Membrane Proteins in the Presence of Phospholipid-Apolipoprotein Particles This example demonstrates that the presence of nanodiscs in prokaryotic in vitro translation systems affects the translation of non-membrane proteins. Show.

An in vitro protein synthesis reaction solution using a plasmid DNA template was prepared as follows. Reactions with standard 50 or 100 μL EXPRESSWAY ™ cell-free expression system (Invitrogen, Carlsbad, CA) were prepared generally according to the manufacturer's instructions and incubated at 37 ° C. The reaction solution contained S30 buffer (2.5 μg Gam protein, 820 U T7 Enzyme, 20 U RNase Out, 0.5 μL ³⁵ S-methionine per mL contained in 0.1% Triton-X100 solution, 600-800 μg of E. coli extract prepared using 1.25 mM amino acid) and 1 × IVPS buffer (58 mM Hepes (pH 7.6), 1.7 mM DTT, 1.2 mM ATP, 0.88 mM UTP, 0.88 mM CTP, 0.88 mM GTP, 34 μg / mL folinic acid, 30 mM acetyl phosphate, 230 mM potassium glutamate, 12 mM magnesium acetate, 80 mM NH ₄ OAc, 0.65 mM cAMP, 30 mM Phosphoenolpyruvate, 2% polyethylene glycol Template DNA 0.5~1μg in call) a (cyclic or linear) was included. The Cycle 3 GFP gene in the vector pCR2.1 was used as a template.

The reaction solution also includes phospholipid-apolipoprotein nanoscale particles comprising membrane scaffold proteins and phospholipids, or US Patent Application Publication No. 2005/0182243 (US Patent Application No. 11), the entire disclosure of which is incorporated herein by reference. (Nanodisc) at a concentration of 1 to 40 μmol (based on protein content) as described in US Pat. PAP was added using a 27 mg / mL PAP stock solution composed of MSP1T2 scaffold protein (US Patent Publication No. 2005/0182243) and DOPC. The reaction was carried out in a 1.5-2 ml Eppendorf microfuge tube in a 30 ° C or 37 ° C thermomixer with moderate shaking (1000-1400 rpm) for 2-6 hours. 30 minutes after the start of the reaction, 1 equivalent of feed solution (based on the initial reaction volume) was added to the reaction. The feed solution contained 57.5 mM HEPES-KOH (pH 8.0), 230 mM potassium glutamate, 14 mM magnesium acetate, 80 mM ammonium acetate, 2 mM calcium chloride and 1.7 mM DTT. The feed solution also contained 1.25 mM of each amino acid (excluding 1.5 mM of methionine), 45 mM glucose 6 phosphate, 0.5 mM NADH, 34 μg folinic acid per mL and 0.65 mM cAMP. . In order to identify the radioisotope of the protein, 2 μL of ³⁵ S-methionine (per 100 μL reaction solution) having a specific activity of 1175 ci / mmole was added to the reaction solution.

  At the end of the incubation, the in vitro protein synthesis reaction was spun at 10,000 × g for a short time, and the supernatant and pellet fractions were loaded separately onto the lane of the SDS-PAGE gel. Specifically, 5 μl of each reaction supernatant was acetone precipitated, pelleted, and suspended in 40 μl of 1 × LDS buffer (Invitrogen, Carlsbad, Calif., Containing 1 mM DTT). 10 μl of this suspension was loaded onto a 4-12% Bis / Tris NuPAGE gel.

  The amount of synthesized GFP and soluble GFP was measured by autoradiography (FIG. 1). FIG. 1a shows autoradiography, which shows that inclusion of phospholipid-apolipoprotein nanoscale particles at 4 μmol and 40 μmol in the translation reaction has no substantially detrimental effect on the yield of non-membrane proteins. It is a bar graph based. FIG. 1 b shows NuPAGE® Novex® 4-12% Bis-Tris gel (Invitrogen, Carlsbad, Calif.) Electrophoresis in the presence (lanes 3 and 4) and absence of 40 μmol PAP. Autoradiography of all translation products (lanes 1 and 3) and soluble translation products (lanes 2 and 4) synthesized below (lanes 1 and 2) are shown. The results show that the presence of phospholipid-apolipoprotein nanoscale particles in the translation reaction does not detectably increase the soluble fraction of non-membrane protein (GFP) synthesized in vitro.

Example 2 In Vitro Synthesis of Membrane Proteins in the Presence of Nanodiscs This example demonstrates the synthesis of solubilized forms of membrane proteins of prokaryotic and eukaryotic origin due to the presence of phospholipid-apolipoprotein particles in an in vitro translation system. It shows that the yield is improved.

³⁵ S-methionine was used to react with EXPRESSWAY ™ cell-free expression system (Invitrogen, Carlsbad, Calif.) Containing 20 μmol of PAP to translate EmrE (bacterial membrane protein, multidrug resistance protein). In vitro protein synthesis reaction was carried out in the same manner as described in Example 1. Total protein and soluble protein obtained from the in vitro synthesis reaction were subjected to electrophoresis as described in Example 1. The results of autoradiography of NuPAGE® Novex® 4-12% Bis-Tris gel (Invitrogen, Carlsbad, Calif.) After subjecting the translation product to electrophoresis are shown in the bar graph of FIG. 2a. The presence of PAP in the in vitro translation mixture increased the yield of soluble EmrE protein by at least a 5-fold.

  The translation product was subjected to NativePAGE (trademark) Novex (registered trade mark) 3 to 12% Bis-Tris gel electrophoresis, and autoradiography was performed to analyze whether or not the synthesized protein exists as a complex. The EmrE protein translated in the absence of PAP did not migrate into the gel, but rather remained at the bottom of the well and was probably aggregated. The EmrE protein synthesized in the presence of 20 or 25 μmol of PAP migrated into the gel and migrated into the higher molecular weight range than in the case of PAP alone (not derived from IVPS reaction). GFP (soluble non-membrane protein) migrated similarly in the native gel regardless of the presence or absence of PAP during synthesis, indicating that it is not incorporated into PAP like EmrE (membrane protein). .

  Furthermore, mammalian membrane protein, human potassium channel subfamily K member 13 protein (Genbank accession no. NM022054, gi16306554, cDNA available from Ultimate ™ ORF clone collection, Invitrogen, 45 kDa with 6 transmembrane domains. Of protein) were reacted using the EXPRESSWAY ™ cell-free expression system described in Example 1 (Invitrogen, Carlsbad, CA) (containing 4 μmol of PAP in the reaction system) and translated in vitro. Using the pEXP3 vector, a template was added to the reaction solution at 700 ng per 100 μL of the reaction solution. Total protein and soluble protein obtained from the in vitro synthesis reaction were subjected to electrophoresis as described in Example 1. The results of autoradiography of NuPAGE® Novex® 4-12% Bis-Tris gel (Invitrogen, Carlsbad, Calif.) After subjecting the translation product to electrophoresis are shown in the bar graph of FIG. 2b. In this case, the amount of soluble membrane protein increased more than twice due to the presence of PAP.

<Example 3> Co-localization of IN VITRO-synthesized membrane protein with phospholipid-apolipoprotein particles In this example, the presence of phospholipid-apolipoprotein particles in an in vitro translation system was synthesized membrane protein. To result in insertion into the PAP.

A His tag was included in apolipoprotein particle protein or scaffold protein (MSP1T2). 20 μmol of PAP prepared with the MSP1T2 His-tagged scaffold protein could be purified using Ni-NTA resin (FIG. 3a, Coomassie stained lanes 5-8, including column elution fraction). As a control, the EmrE protein was subjected to a cell-free translation reaction containing ³⁵ S-methionine in the absence of PAP using the EXPRESSWAY ™ cell-free expression system described in Example 1 (Invitrogen, Carlsbad, Calif.). It was made to synthesize. EmrE (without His tag) was not purified with Ni-NTA resin (Figure 3b, lanes 5-8, including column elution fractions). However, EmrE (which co-purifies with PAP phospholipid-binding protein) was purified with Ni-NTA resin by adding PAP with His-tagged modified apolipoprotein protein to the reaction (FIG. 3c, lane 5- 8 includes column eluent fraction). That is, it shows that EmrE was inserted into PAP having a purification tag.

  The results were evaluated by analysis of a Native Blue gel, and the radioisotope-identified bacterial membrane protein EmrE (loaded with about 0.3 μg protein) expressed without PAP was aggregated at the top of the gel. It became clear (Fig. 5a, lane 2, autorad). However, when PAP was added to the expression reaction, EmrE formed a complex (Fig. 4a, lanes 3, 4, autorad) that migrated into the gel and migrated with a higher molecular weight than PAP (Fig. 4b, lane). J, Coomassie stained gel). GFP (non-membrane protein) showed the same molecular weight with or without the addition of PAP to the translation system (FIG. 4a, lanes 5 and 6, respectively).

<Example 4> A membrane protein synthesized in vitro using a His-tagged nanodisk can be purified with Ni-NTA resin. In this example, the presence of a nanodisk in an in vitro translation system was synthesized using a tagged nanodisk. It shows that the purified membrane protein can be purified.

The GFP and MscL genes (bacterial mechanosensitive channel (17 kDa) membrane protein) were cloned into the pEXP4 vector. The C-terminus of the expressed protein was not His-tagged because both genes contained a stop codon. EXPRESSWAY ™ cell-free expression system (Invitrogen, Carlsbad, CA) containing ³⁵ S-methionine and using 1 μg of GFP and MscL template was added with PAP (40 μmol) containing His-tagged scaffold protein (or Not added). After incubation, the reaction solution was loaded onto a Ni-NTA column.

  The results of column purification are shown in FIG. 5 (L = load, FT = flow through, W = wash, E1-E4 = elution). Regardless of the presence of the Nanodisc translation reaction (FIGS. 5a and 5b), GFP (not a membrane protein) cannot be purified using Ni-NTA columns. MscL was shown to be purified on Ni-NTA columns only when Nanodiscs were included in the translation reaction (FIGS. 5c and 5d). That is, MscL is inserted into PAP.

<Example 5> A membrane protein synthesized in vitro using a nanodisk is associated with the nanodisk, and the solubility is improved. In this example, the solubility is improved by the presence of phospholipid-apolipoprotein particles in an in vitro translation system. It is shown that a synthesized membrane protein is synthesized and inserted into PAP to improve solubility.

Using the pEXP3 vector included in the EXPRESSWAY ™ cell-free expression system containing ³⁵ S-methionine (Invitrogen, Carlsbad, Calif.), The bacterial membrane protein EmrE and the mammalian ORFs “IOH5384” (human plasma membrane proteolipid (plasmolipin)) Encoding) and "IOH22669" (encoding human adrenomedullin receptor (ADMR)). An aliquot of the total protein and soluble fraction from the in vitro synthesis reaction was run on a NuPAGE® Novex® Bis-Tris gel (Invitrogen, Carlsbad, Calif.), Resulting in the addition of PAP to the in vitro synthesis reaction Occasionally, improved solubility of bacterial and mammalian membrane proteins was shown, but GFP solubility was not affected by the presence or absence of PAP in the in vitro synthesis reaction (FIGS. 6a and 6b).

  In the autoradiography of the blue native gel of FIG. 6c, bacterial membrane protein EmrE, mammalian ORFsIOH 5384 (encoding human plasma membrane proteolipid (plasmolipin)), and IOH22669 (encoding human adrenomedullin receptor (ADMR)) are PAP. Indicates that it will be inserted. When PAP was added to the reaction, the radioisotope-identified EmrE, IOH 5384, and IOH22669 shifted upward. GFP (non-membrane protein) showed the same molecular weight with or without the addition of PAP to the in vitro synthesis reaction.

Example 6 LUMIO Detection of Inserted Membrane Protein The gene for EmrE (bacterial membrane protein) was cloned into an N-terminal vector containing the LUMIO ™ tetracysteine motif tag (pEXP6, Invitrogen, Carlsbad, Calif.). The EmrE construct did not contain a His tag. PAP (40 μm) was added or not added to the reaction solution of the EXPRESSWAY ™ cell-free expression system (Invitrogen, Carlsbad, Calif.), And after the synthesis, the reaction solution was loaded onto a Ni-NTA column (L = Load, FT = flow through, W = wash, E1-E4 = elution). LUMIO ™ detection reagent (Invitrogen, Carlsbad, CA) was added to the 4-12% NuPAGE® Bis- according to the manufacturer's instructions of the LUMIO ™ Green in-gel detection kit (Invitrogen, Carlsbad, CA). Prior to analysis of the tris gel, it was added to the sample and the gel was imaged with a phosphorimager. In FIG. 7a, the translation reaction solution containing PAP was analyzed. The LUMIO ™ sequence (EmrE translation product) in the fraction eluted from the Ni-NTA column (purification using the His tag of the PAP scaffold protein) was detected. In FIG. 7b, the translation reaction solution containing no PAP was analyzed. In the elution fraction from the Ni-NTA column, no LUMIO ™ sequence (EmrE translation product) was detected. From the above, the membrane protein can be incorporated into PAP and synthesized in vitro in a solubilized form, and can be efficiently purified using the tag on the apolipoprotein of PAP.

<Example 7> A luciferase protein was expressed in a cell-free CHO cell extract containing nanodisks and containing no eukaryotic in vitro protein synthesis reaction PAP or containing 0.1 to 19 μmol of PAP. RNA was prepared from the pEXP4 vector containing the luciferase gene using mMMessageMachine (AMBION). 6 μg of RNA was used in the translation reaction.

CHO cell extracts were prepared according to the following protocol.
Measure cell number / viability.
1. Centrifuge gently (10 min, 800-1000 rpm) and collect cells.
2. Add 4 mM DTT to Buffer A.
3. Wash cells with 250 mL of buffer A (very gentle; do not completely resuspend cells).
4). Wash cells with 250 mL buffer A (just add buffer, do not resuspend cells).
5. Resuspend pellet in half-pellet volume of buffer A (+1 mM PMSF).
6). Sample aliquots for cell counting.
7). Pass through a 100 psi French press.
8). Sample aliquots for cell counting.
9. Measure cell number / viability of both aliquots. In the first aliquot most cells must be normal. In the second aliquot, the cells must be <20% viable.
10. Centrifuge at 15 min x 14000 rpm (operate with microcentrifuge).
11. The supernatant is collected (the pellet is stored at −80 ° C. for further use).
12 Add 1 mM CaCl ₂ , 0.15 U / μl of Micrococcus derived nuclease.
13. Incubate for 5 minutes at RT.
14 The reaction is stopped by adding 2 mM EGTA.
15. A 50-80 μl sample is taken, rapidly frozen in liquid N ₂ and stored at −80 ° C.
16. _{A 260} and _{A 280} of the supernatant (1/200 dilution). Measure. Must be> 100 units.
Buffer A
40 mM Hepes KOH (pH 7.8),
100 mM KOAc,
4 mM magnesium (OAc) 2,
4 mM DTT (uses just after preparation).
5 mg / ml (0.5 μl) creatine kinase, Buffer # 2 Proteios wheat germ system (1.5 μl), RNaseOut (0.25 μl), Buffer # 1 (0.85 ul), Smet (0.5 μl) and BHK extract (6 μl) was used for translation. The translation reaction was incubated at 33 ° C. for 1 hour. 2.5 μl of each translation reaction solution was used for luciferase analysis.
After completion of the protein synthesis reaction, luciferase activity was detected. As shown in FIG. 8, the presence of PAP did not show a detrimental effect on the protein synthesis of the CHO cell extract.
Transcription-translational expression of bacterial membrane protein EmrE, human ORF 21132, vesicle-associated calmodulin-binding protein, human ORF 21140 cyclin-dependent kinase 2 (CDK2) and luciferase with rabbit reticulocyte lysate containing ³⁵ S-methionine Using the system (Promega), translation was performed according to the manufacturer's instructions. In one reaction set, 1.25 μL of 27 mg / mL PAP was added into the synthesis system. In the second reaction set, no PAP was added into the synthesis system. The translation product was subjected to Blue Native gel electrophoresis and autoradiography was performed. FIG. 9 shows that in the case of the membrane protein EmrE, an increase in solubility (the radioisotope-identified protein product enters the gel and migrates) was seen in the presence of PAP. This was not the case with the non-membrane proteins human ORF 21132 (vesicle-associated calmodulin-binding protein), human ORF 21140 cyclin-dependent kinase 2 (CDK2) and luciferase. FIG. 10 shows that the same results were obtained using CHO cell extracts.

<Example 8> Improvement of solubility of membrane protein when co-expressed with apolipoprotein in in vitro synthesis reaction This example is based on the presence of apolipoprotein construct in an in vitro translation system that does not contain PAP. It shows that the synthesis of membrane protein is promoted.

  In separate tests, bacterial and mammalian membrane proteins were transcribed and translated from plasmid constructs in a cell-free synthesis system. In one test set, the gene encoding the bacterial membrane protein EmrE was cloned into the vector pEXP5-NT to produce a protein of interest (POI) construct. In another test set, the gene encoding the human membrane protein GABA A receptor (Invitrogen ULTIMATE ™ ORF collection clone IOH10885 (Genbank accession no. BC022449, gi184490266) was cloned into the vector pEXP5-NT and the target protein or “ In both experiments, human apolipoprotein A1 (Invitrogen ULTIMATE ™ ORF Collection Clone IOH7318, Genbank accession no. NM00839, a construct encoding gi4557320, pEXP3-Apol, was synthesized in several in vitro experiments. As a result, Apo A1 is the target membrane protein. Expressed in the same reaction system.

An in vitro protein synthesis reaction using a plasmid DNA template was prepared as follows: a standard 100 μL EXPRESSWAY ™ reaction was prepared and basically instructed by the manufacturer (Invitrogen, Carlsbad, Calif.). And incubated at 37 ° C. 1 μg of DNA construct was added to each of the reactions. Reactions were performed at 30 ° C. or 37 ° C. in a 1.5-2 ml microfuge tube in an Eppendorf Thermomixer with moderate shaking (1000-1400 rpm) for 2-6 hours. Thirty minutes after the start of the reaction, 1 equivalent of feed solution (based on the first reaction volume) was added to the reaction according to the manufacturer's instructions (Invitrogen Expressway manual, Invitrogen, Carlsbad, CA). In order to identify the protein as a radioisotope, ³⁵ S-methionine was added to the reaction solution.

  The reaction was carried out in the presence or absence of pEXP3-Apol for each target membrane protein. Furthermore, each reaction set was carried out in the presence or absence of phospholipid (DMPC for 100 μg / mL for EmrE translation reaction, DPPC for 100 μg / mL for GABA A receptor translation reaction).

  After the incubation, the in vitro protein synthesis reaction was spun at 10,000 × g for a short time, and the supernatant and pellet fractions were loaded separately onto the lane of the SDS PAGE gel. 5 μl of the supernatant of each reaction was acetone precipitated, pelleted, suspended in 40 μl (Invitrogen) containing 1 × LDS buffer (containing 1 mM DTT), 10 μl of which was 4-12% Bis-Tris NuPAGE (registered trademark). ) Loaded on the gel (Figure 11).

  As a result of the test, the presence of the Apo A1 construct in the translation reaction increased the yield of soluble EmrE (lanes 7 and 8, FIG. 11a) compared to the translation in the absence of the Apo A1 construct (lanes 5 and 6). ). When Apo A1 also lacks the Apo A1 construct (lanes 5 and 6) which greatly improves the soluble yield of GABA A (lanes 7 and 8, FIG. 11b) when compared to the soluble yield. Autoradiography also demonstrated that apolipoprotein A1 itself is expressed in a solubilized form (lanes 3 and 4 in FIGS. 11a and 11b).

Example 9 In Vitro Synthesis of Membrane Protein in the Presence of Apolipoprotein This example shows that the presence of apolipoprotein in an in vitro translation system improves the synthesis yield of solubilized membrane protein.

EmrE (bacterial membrane protein) was translated using ³⁵ S-methionine in the EXPRESSWAY ™ in vitro synthesis system (Invitrogen, Carlsbad, Calif.) As described in the previous examples. Translation was performed with 2.5-15 μg of Apo A1 protein. Total and soluble proteins prepared by in vitro synthesis were subjected to gel electrophoresis and autoradiography was performed. As a result, the production amount of Apo A1 protein was increased by the in vitro synthesis reaction, and the production amount of soluble membrane protein was increased (FIG. 12a).

  In addition, mammalian membrane proteins (human GABA A receptor protein) were translated with an in vitro translation system containing 2.5-15 μg of Apo A1 protein. Again, the presence of Apo A1 protein in the translation system significantly increased the amount of soluble membrane protein (FIG. 12b).

Example 10 Association of Apolipoprotein and Translated Membrane Protein in In Vitro Synthesis System In this example, the presence of Apo A1 protein in the in vitro translation system (a membrane protein is synthesized) indicates that the synthesis of soluble membrane protein. This indicates that Apo A1 associates with the translated protein. This example also shows that when apolipoprotein is present, the solubility of the membrane protein is usually maintained in the presence of the surfactant, even in the absence of the surfactant.

  The Expressway in vitro translation reaction was performed in a total volume of 100 μL. Using the EmrE gene as a nucleic acid template, cloning was performed in frame in pEXP5-NT (Invitrogen, Carlsbad, CA) encoding a His tag at the N-terminus. Apo A1 purified from human plasma was included in the in vitro synthesis reaction. After performing an in vitro synthesis reaction according to the manufacturer's instructions, the in vitro synthesized EmrE (with His tag) was purified on a Ni-NTA column by gravity flow using 20 mM Tris (pH 7.5), 200 mM NaCl. Dodecyl maltoside surfactant (usually added to the buffer to maintain EmrE solubility) was omitted.

  EmrE protein was eluted using 1M imidazole in the same Tris-NaCl buffer. The Coomassie gel shown in FIG. 13a shows that untagged Apo A1 protein (26 kDa) was co-purified with His-tagged EmrE (M is protein molecular weight marker, L is loaded fraction, FT is flow-through) , W1 and W2 are a series of column washes, E1-E4 are a series of elution fractions). It is confirmed by autoradiography that the EmrE protein is eluted in the same fraction as the purified Apo A1 protein (FIG. 13b), that is, shows a physical association between the proteins.

a) is a bar graph representing GFP produced by IVPS reaction in the presence (right column of each pair) and absence (left column of each pair) of 4 μmol and 40 μmol of PAP. b) autorad representing GFP produced in the IVPS reaction in the presence and absence of PAP. a) Bar graph showing total and soluble expression of bacterial EmrE in the presence and absence of 20 μmol PAP. b) Bar graph showing total and soluble expression of mammalian potassium channel protein in the presence and absence of 4 μmol PAP. a) represents a gel of PAP obtained by purifying a modified apolipoprotein fused with a His tag with Ni-NTA resin and staining it with Coomassie. Lanes 5-8 are the elution fractions. b) represents a Coomassie stained gel for translation of EmrE protein (no PAP), elution after binding to Ni-NTA resin. Lanes 5-8 are elution fractions c) represent a Coomassie stained gel of EmrE protein translated using PAP. The modified apolipoprotein fused with the His tag was purified with Ni-NTA resin. Lanes 5-8 are the elution fractions. EmrE protein translated in the presence of PAP (lanes 3 and 4) and in the absence of PAP (lane 2), and GFP expressed in the presence (lane 6) or absence (lane 5) of PAP Autoradiography. Translation product of GFP in the presence and b) absence of PAP with his-tagged modified apolipoprotein, and c) presence and d) absence of PAP with his-tagged modified apolipoprotein 2 shows gel autorads showing the purification of the translation product of MscL in Ni-NTA by Ni-NTA. Figure 2 shows gel autorads of GFP translation products (a and b) translated in the presence of PAP and EmrE translation product (c) translated in the presence of PAP. a) shows the detection of EmrE lumino by the lumino sequence synthesized in the translation reaction containing PAP. b) shows EmrE lumino detection by the lumino sequence synthesized in the translation reaction that did not contain PAP. The bar graph which shows the luciferase activity at the time of carrying out the translation reaction of luciferase by increasing the quantity of PAP. Native gel autoradiography of rabbit reticulocyte translation reaction product with or without PAP. Native gel autoradiography of rabbit reticulocyte translation reaction product with or without PAP. The autoradiography of the gel after subjecting the translation product of an in vitro protein synthesis reaction to electrophoresis, with or without Apo A-I, is shown. (A) In the in vitro protein synthesis reaction, the overall yield of bacterial EmI protein is not affected in the presence of apolipoprotein or phospholipid (lanes 1-4), while the yield of soluble EmrE protein is the presence of apolipoprotein. Enhanced below (lanes 5-8). (B) In the in vitro protein synthesis reaction, the overall yield of mammalian GABA A protein is not affected in the presence of apolipoprotein or phospholipid (lanes 1-4), while the yield of soluble GABA A protein is Enhanced in the presence of protein (lanes 5-8). The autoradiography of a gel when an in vitro protein synthesis reaction was carried out in the absence of Apo A-I or under a condition in which the content of ApoA-I was changed and the translation product was subjected to electrophoresis. (A) In the in vitro protein synthesis reaction, the overall yield of bacterial EmrΕ protein is not affected in the presence of apolipoprotein (lanes 1-4), while the yield of soluble EmrE protein is enhanced in the presence of apolipoprotein. (Lanes 5-8). (B) In the in vitro protein synthesis reaction, the total yield of mammalian GABA A protein is not affected in the presence of apolipoprotein (lanes 1-4), while the yield of soluble GABA A protein is the presence of apolipoprotein. Enhanced below (lanes 5-8). In an in vitro protein synthesis reaction containing Apo A-I, the his tag was fused, and the ³⁵ S-labeled EmrE translation product was isolated on a Ni-NTA column, and then subjected to electrophoresis. Autoradiography is shown. (A) Column fraction shows co-elution of Apo A1 and EmrE. (B) The presence of EmrE in the eluted fraction was confirmed by autoradiography.

Claims (20)

  A membrane protein synthesis method comprising adding a nucleic acid template to an in vitro protein synthesis system including a cell extract and an apolipoprotein, and incubating the nucleic acid template and the in vitro protein synthesis system to synthesize a membrane protein in a solubilized form A method comprising:   The method according to claim 1, wherein the membrane protein is a transmembrane protein, an integral membrane protein, or a peripheral membrane protein.   The method of claim 1, wherein the membrane protein is synthesized in a solubilized form.   The method of claim 1, wherein the cell extract is a prokaryotic cell extract.   The method according to claim 4, wherein the cell extract is an E. coli cell extract.   The method of claim 1, wherein the cell extract is a eukaryotic cell extract.   The cell extract is a wheat germ extract, a Drosophila embryo extract, a rabbit reticulocyte extract, a scallop extract, a mouse brain extract, a chicken chick brain extract or an extract from cultured cells. the method of.   The method of claim 1, wherein the nucleic acid template is an RNA template.   The method of claim 1, wherein the nucleic acid template is a DNA template.   The method of claim 1, wherein the apolipoprotein is present in a phospholipid-apolipoprotein particle.   The method of claim 1, wherein the apolipoprotein is a natural apolipoprotein, a variant of a natural apolipoprotein, or a modified apolipoprotein.   The method according to claim 14, wherein the apolipoprotein is apolipoprotein A1 or a variant thereof.   The method of claim 1, wherein the apolipoprotein is a modified apolipoprotein comprising one or more amphipathic helical domains.   The method of claim 1, wherein the apolipoprotein comprises one or more amino acid sequence tags.   15. The method of claim 14, wherein the one or more amino acid sequence tags are His tags.   15. The method of claim 14, further comprising purifying the membrane protein using an apolipoprotein sequence tag.   In vitro protein synthesis system comprising a cell extract, one or more energy sources and an apolipoprotein   18. The in vitro protein synthesis system according to claim 17, wherein the cell extract is a prokaryotic cell extract.   The in vitro protein synthesis system of claim 17, wherein the in vitro synthesis system comprises a cell extract of E. coli.   18. The in vitro protein synthesis system according to claim 17, wherein the cell extract is a eukaryotic extract.

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