WO2023235921A1 - Procédés de production de variants polypeptidiques - Google Patents

Procédés de production de variants polypeptidiques Download PDF

Info

Publication number
WO2023235921A1
WO2023235921A1 PCT/AU2023/050493 AU2023050493W WO2023235921A1 WO 2023235921 A1 WO2023235921 A1 WO 2023235921A1 AU 2023050493 W AU2023050493 W AU 2023050493W WO 2023235921 A1 WO2023235921 A1 WO 2023235921A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
cells
cell
variant
detergent
Prior art date
Application number
PCT/AU2023/050493
Other languages
English (en)
Inventor
Daniel Scott
Christopher DRAPER-JOYCE
Riley CRIDGE
Original Assignee
The Florey Institute Of Neuroscience And Mental Health
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2022901541A external-priority patent/AU2022901541A0/en
Application filed by The Florey Institute Of Neuroscience And Mental Health filed Critical The Florey Institute Of Neuroscience And Mental Health
Publication of WO2023235921A1 publication Critical patent/WO2023235921A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B10/00—Directed molecular evolution of macromolecules, e.g. RNA, DNA or proteins
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/06—Methods of screening libraries by measuring effects on living organisms, tissues or cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

  • This disclosure relates generally to an improved method for selection or directed evolution of proteins, and their encoding sequences, to engineer proteins having desired traits.
  • Eukaryotic cells express a large variety of proteins that are responsible for mediating extracellular stimuli and interaction I cell-cell signaling, which in turn involve many complex protein interactions intracellularly.
  • proteins found at the cell membrane respond to extracellular stimuli, triggering downstream signaling pathways that involve other interactions with intracellular proteins, which can be located in the cytoplasm, mitochondria, the nucleus, the endomembrane structures (nuclear membrane, golgi apparatus and endoplasmic reticulum), or other intracellular organelles.
  • Many of these proteins, in particular the membrane proteins are involved in physiological disease or disorders and are important drug targets.
  • GPCRs G protein-coupled receptors
  • a major bottleneck for studying the structure of proteins and drug discovery is sample preparation.
  • Membrane proteins are particularly challenging as they tend to be very lowly expressed and have complex, dynamic structures due to their location at the membrane (having functional extracellular, intracellular and/or transmembrane interfaces). These factors constrains protein yield and sample viability.
  • target proteins frequently need to be detergent solubilized such that they can be purified under aqueous conditions. This is particularly challenging for membrane proteins, as often there is an insufficient window between the concentration of detergent required to effectively solubilize the membrane protein but not cause aggregation or misfolding, such that this critical step prevents further downstream purification.
  • attempts to address such challenges have involved complexing such proteins with stabilizing ligands, inserting stabilizing mutations, or reconstitution of the proteins in stabilizing detergents or in the case of membrane-proteins, reconstitution on membrane scaffolds.
  • Low expression of target proteins can sometimes be addressed by use of fusion proteins and/or the introduction of expression-boosting mutations. For example, systematic mutagenesis has been used to select for increased receptor expression in Escherichia coli.
  • the target protein is expressed in a sub-cellular localization that makes screening for functional activity (e.g. ligand binding, activation, partner-protein binding or signaling capacity) of the target protein difficult or inefficient, due to the difficulty in allowing access of screening agents to the target cell.
  • functional activity e.g. ligand binding, activation, partner-protein binding or signaling capacity
  • Directed evolution is a powerful tool for generating nucleic acids and molecules they encode with specific properties, such as improved, altered or novel characteristics and/or functions for a variety of industrial, therapeutic and research applications.
  • proteins may be selected for improved or altered solubility, expression level, pH stability, thermostability, detergent stability, organic solvent stability, folding properties, binding characteristics or improved performance such as improved enzymatic performance.
  • the critical step in all directed evolution methods is the screening and selection of protein libraries for desired phenotypes.
  • Selection techniques allow the examination of very large libraries by linking the phenotype of a protein to its genotype, allowing rapid identification of interesting variants.
  • Selection methods such as phage display, yeast display, bacterial display, mRNA display and ribosome display are well established and routinely used to identify interesting biomolecules from large libraries, however none of these methods have been demonstrated to be suitable to the selection of mammalian proteins that depend on complex intracellular synthesis or processing steps, or the presence of intracellular structures for their proper folding, stability or functional integrity. In many cases, these process are highly laborious, and the selected proteins often fail to recapitulate the normal structure and/or function of the native protein.
  • variant proteins having a desired activity can be efficiently engineered or selected from a large possible assortment of variant sequences. This involves exposing cells expressing the variant proteins to a detergent or detergent-like compound, which advantageously allows permeabilised cells that express favorable protein mutants to be identified and isolated. Further, the present disclosure is predicated on the inventors' surprising discovery that detergent-stable, functional variant membrane proteins can be selected for enhanced expression in mammalian cells, by means of exposing cells expressing the variant membrane protein to a sub-critical solubilisation concentration of detergent or detergent-like compound.
  • the present disclosure provides a method for selecting a variant of a protein of interest having a desired activity from a library of expressed nucleic acid sequences encoding variants of the protein of interest, the method comprising: i. providing a population of mammalian cells, wherein each cell of the population of cells comprises and expresses no more than two nucleic acid sequences of said library;
  • the desired activity of the selected variant is increased cellular expression; altered affinity for the labelled agent (increased or decreased affinity); altered protein-protein interaction with a target interacting protein; a stabilised conformational state; modified protein trafficking to the cell membrane; modified protein trafficking to the cell nucleus; and/or modified protein trafficking to the mitochondria, and/or a longer protein half-life when compared to the protein from which the variant is derived and/or the corresponding wild-type protein.
  • the desired activity of the selected variant is compared to the wildtype protein.
  • the protein of interest is a membrane protein. In another embodiment, the protein of interest is an intracellular protein. [0017] In another embodiment, the exposed cells retain cell viability.
  • the method further comprises purifying the variant of a protein of interest from the selected cell.
  • the present disclosure provides a method for selecting a detergent-stable functional variant of a membrane protein of interest from a library of expressed nucleic acid sequences encoding variant membrane proteins, the method comprising:
  • each cell of the population of cells comprises and expresses no more than two nucleic acid sequences of said library
  • sub-CSC sub-critical solubilisation concentration
  • the method further comprises recovering the selected cells and repeating steps ii-iv.
  • the selected detergent-stable functional variant membrane protein has increased cellular expression when compared to the protein from which the variant is derived.
  • the protein from which the variant is derived is a wildtype protein.
  • the protein of interest or membrane protein of interest is located in the cell membrane or an intracellular membrane of the mammalian cell.
  • the detergent is selected from neopentyl glycol (NG) detergents and maltoside detergents.
  • the NG detergent is LMNG.
  • the maltoside detergent is an alkyl maltoside.
  • the alkyl maltoside is DDM.
  • the detergent or detergent-like compound is a saponin.
  • the detergent or detergent-like compound may be selected from digitonin, diosgenin, glycol-diosgenin (GDN), or other saponins.
  • the methods disclosed herein further comprise isolating from the selected cell the nucleic acid sequence encoding the variant protein having the desired activity or the detergent-stable variant membrane protein; and transducing the nucleic acid sequence encoding the variant protein having the desired activity or the detergent-stable variant membrane protein into a population of mammalian cells, and repeating steps ii-iv.
  • the methods disclosed herein further comprise: isolating from the selected cell, the nucleic acid sequence encoding the variant protein having the desired activity or the detergent-stable variant membrane protein; generating one or more mutations in the isolated sequence to produce a second library of nucleic acid sequences encoding variant proteins having the desired activity or detergent-stable variant membrane proteins; introducing said library of nucleic acid sequences encoding variant proteins having the desired activity or detergent-stable variant membrane proteins into a second population of cells to provide a population of mammalian cells, wherein each cell of the population of cells comprises and expresses no more than two nucleic acid sequences of said second library, and repeating steps ii-iv.
  • the one or more mutations in the isolated nucleic acid sequence are generated by random mutagenesis. In another embodiment, the one or more mutations in the isolated nucleic acid sequence are rationally designed.
  • the selected variant protein having the desired activity or selected detergent-stable variant membrane protein is expressed at a higher level when compared to the corresponding variant protein from which it was derived.
  • the nucleic acid sequence encoding variant proteins having the desired activity or detergent-stable variant membrane proteins is stably integrated into the genome of the cell.
  • the library of nucleic acid sequences is introduced into the population of cells using a viral vector.
  • the viral vector is an adenoviral vector, an adeno-associated viral vector or a lentiviral vector.
  • the viral vector is a lentiviral vector.
  • the lentiviral vector comprises one or more of: a. HIV-1 5' and 3' long terminal repeats; b. a psi packaging signal; c. a cloning site; d. a Cytomegalovirus promoter; e. an IRES; and f. one or more selectable markers.
  • the selectable marker is a gene encoding a fluorescent protein, a molecular marker gene, an antibiotic resistance gene or SacB gene.
  • the agent is a ligand, such as an antibody, antibody fragment or nanobody, or a sensor of the membrane protein of interest.
  • the agent is the agent is a nanobody, a G protein or a mini G protein.
  • the agent in the contacting step is labelled with a fluorescent label, an isotope, magnetic particles or a dye.
  • the selection step comprises cell sorting. In certain embodiments, the selection step comprises one or more consecutive rounds of cell sorting. In one embodiment, the selection step comprises 2-20 consecutive rounds of cell sorting. In another embodiment, the cell sorting is fluorescence activated cell sorting (FACs).
  • FACs fluorescence activated cell sorting
  • the selected cell is cultured to expand the number of cells.
  • the method further comprises purifying the variant of a protein of interest from the selected cell.
  • the methods disclosed herein further comprise purifying the detergent-stable functional variant membrane protein from the selected cell.
  • the membrane protein of interest is an integral membrane protein.
  • the variant protein of interest or the variant membrane protein is the integral membrane protein, a receptor protein, an ion channel, a tyrosine kinase or a transporter.
  • the variant protein of interest or the variant membrane protein is a G-protein coupled receptor (GPCR).
  • GPCR G-protein coupled receptor
  • the GPCR is selected from a group consisting of neurotensin receptors, dopamine receptors, serotonin receptors, vasopressin receptors and adrenergic alpha 1A (alA) receptors.
  • alA adrenergic alpha 1A
  • the GPCR is a chimeric GPCR.
  • the present disclosure provides a method for selecting a variant of a chimeric GPCR of interest from a library of expressed nucleic acid sequences encoding variant chimeric GPCRs, wherein the chimeric GPCR comprises an Nb6 binding epitope, the method comprising:
  • each cell of the population of cells comprises and expresses no more than two nucleic acid sequences of said library; ii. exposing the population of cells to a detergent or a detergent-like compound at a concentration sufficient to permeabilise the cell membrane, while retaining the genetic integrity of the exposed cell; ill. contacting the exposed cells with a labelled Nb6 to facilitate detection of a conformational state of the chimeric GPCR encoded by the nucleic acid sequences of said library; and subsequently iv. selecting from the population of cells, a cell expressing a variant chimeric GPCR as a function of the detected conformational state, wherein the selected cell comprises a detergent-stable functional variant chimeric GPCR.
  • the detergent or detergent-like compound is digitonin or
  • the chimeric GPCR comprises (an Nb6 binding epitope).
  • the Nb6 binding epitope comprises the intracellular ends of transmembrane 5, the intracellular ends of transmembrane 6, and/or the intracellular loop 3 domain from the kappa opioid receptor (KOR).
  • the Nb6 binding epitope comprises the amino acid sequence of any one of SEQ ID NOs: 15-21.
  • the Nb6 binding epitope comprises the amino acid sequence of SEQ ID NO: 15.
  • the present disclosure provides a method for selecting a variant of a chimeric GPCR of interest from a library of expressed nucleic acid sequences encoding variant chimeric GPCRs, wherein the chimeric GPCR comprises a G protein binding site or a mini G protein binding site, the method comprising: i. providing a population of mammalian cells, wherein each cell of the population of cells comprises and expresses no more than two nucleic acid sequences of said library; ii. exposing the population of cells to a detergent or a detergent-like compound at a concentration sufficient to permeabilise the cell membrane, while retaining the genetic integrity of the exposed cell; ill.
  • the detergent or detergent-like compound is digitonin or
  • the mammalian cell is a mouse cell, a rat cell, a CHO cell, an African Green Monkey cell or a human cell. In one embodiment, the mammalian cell is a human cell.
  • Figure 1 provides a diagrammatic example of mammalian cell-based directed evolution method with a detergent treatment step according to an embodiment of the invention. Sequence randomisation-. Error prone PCR was used to generate a diverse library of a gene of interest.
  • Generate lentivirus The gene library was ligated into a customized lentiviral transfer plasmid featuring an IRES mCherry. Cells were transfected with the following 3 rd generation lentiviral components - customized transfer plasmid, packaging plasmids, and envelope plasmid, and lentivirus-containing media was harvested. Transduce cells. Cells were transduced with librarycontaining lentivirus at a MOI of ⁇ 1 (approximately 30% transduced).
  • Permeabilize cells and treated with fluorescent ligand The transduced cell culture was treated with detergents and incubated with fluorescent ligand to label functional protein mutants encoded by favourable gene mutants from the library.
  • Sort for high expression Treated cells were subjected to FACS to select cells containing favourable library-encoded mutants. Multiple rounds of selection are performed in succession to enrich the desired population.
  • Isolate DNA The library gene pool of the selected population was isolated via genomic extraction and PCR and individual mutants were isolated for characterisation. The library gene pool can be used as the template for the next generation.
  • Figure 2 is a diagrammatic representation of the viral cassette of lentiviral transfer plasmid pLenti-displ-sacB (SEQ ID NO: 1) based on lentiviral transfer plasmid pCSC (Ngo et al., 2020). Viral components flank the transgene.
  • Figure 3 shows the use of detergents at (A) sub-critical solubilizing concentration
  • CSC and (B)equal-to-or-above CSC (>CSC).
  • B depicts the labelling of intracellularly located receptor, i.e. present in organelle membranes, with the use of detergent-like compounds such as digitonin, diosgenin, GDN, or other saponins.
  • FIG. 4 depicts experiments determining cell tolerance and finding sub-CSC of common detergents before selections.
  • A) HEK293F cells were treated with increasing concentrations of detergent, either DDM or LMNG and analysed for their forward-scatter (FSC-A) and side-scatter (SSC-A) profile using flow cytometry.
  • FSC-A forward-scatter
  • SSC-A side-scatter
  • HEK gate signifies particles with cell like properties.
  • C) Cells within the HEK gate from selected detergent conditions were analysed for cell death using nuclear stain DAPI using flow cytometry.
  • Figure 5 shows the differential effects of sub-CSC concentrations of detergents on a prototypical pair of GPCR mutants.
  • Treatment of HEK 293-F cells expressing two different mutants of the neurotensin receptor (NTS1) were treated with sub- concentrations of either detergent (A) LMNG or (B) DDM for 10 min at room temperature.
  • Receptor competency can be tracked by assaying fluorescence ligand binding with flow cytometry, revealing mutant two is more sensitive to detergent than mutant 1.
  • Figure 6 shows experiments detecting intracellular pools of functional receptor protein with addition of permeabilizing detergent.
  • HEK293F cells transduced with D2 or 5-HT2A receptors using pLenti-displ lentivirus were treated with 0.005% digitonin, followed by incubation with 40 nM fluorescent antagonist L0002RED to label intracellular receptor.
  • Cells were analysed using flow cytometry.
  • a and B are heatmap scatterplots displaying forward-scatter (x-axis, cell size) and side-scatter (y-axis, cell complexity) profiles of cells with and without treatment of 0.005% digitonin. Black circles indicate the population of particles that were gated for analysis in Figures 3C-F.
  • Digitonin treated cells exhibit a reduced forward-scatter profile, indicating a reduction in size, but remain a distinguishable and selectable population of events from non-cell particles.
  • Figures 3C-F are histograms of A647 fluorescence of cells, representing binding of fluorescent ligand L0002RED. Cells treated with fluorescent ligand only are shown in purple, while non-specific binding (NSB) control cells treated with additional 10 pM spiperone as competition are shown in black. There was no specific binding of L0002RED at the cell surface for whole cells expressing D2 (C), while a small population of digitonin-treated D2 cells exhibited specific binding (D).
  • FIG. 7 shows FACS analysis of 293-F cells transduced with pLenti-displ-VlA lentivirus.
  • 293-F cells were treated with undiluted pLenti-displ-VlA lentivirus-containing media.
  • the pLenti-displ-VlA culture was treated with fluorescent ligand A647-PhAcALVP (with SR49059 as competitor) and analysed using FACS. Analysis of lentivirus-treated cells for mCherry fluorescence.
  • mCherry FI fluorescence intensity
  • Figure 8 shows 293-F cells treated with dilutions of pLenti-displ-ViA-EPl lentivirus.
  • 293-F cell cultures were treated with various dilutions of VIA EPl library-containing lentivirus to achieve a desired multiplicity of infection (MOI).
  • MOI multiplicity of infection
  • a low MOI of 0.2-0.3 was desired to minimise the probability of co-transductants - cells transduced with multiple virions - which would require a transduction efficiency of 20-26%.
  • Histogram plots are shown of 293-F cultures, which were analysed for 600 - 620 nm fluorescence intensity (mCherry FI) using flow cytometry. mCherry positive (mCherry+) and negative (mCherry-) populations are also highlighted. Cell cultures are labelled by the virus dilution factor, with increasing dilutions of virus shown in progressively darker shades of red.
  • Figure 9 depicts the directed evolution of high-expressing VIA variants.
  • A FACS histograms of populations of cells expressing un-mutated VIA (VIA wt, black) or selected error- prone libraries of VIA (EP1AS3 (red); EP2AS4 (purple) treated with VlA-specific fluorescent peptide, A647-PhAcALVP.
  • Non-specific binding (NSB) is depicted by the blue dashed histogram, determined by treating the VIA EP2AS4 cell population with A647-PhAcALVP and excess unlabeled PhAcALVP as a competitor.
  • B FACS histograms of populations of cells expressing un-mutated VIA (VIA wt, black) or selected error- prone libraries of VIA (EP1AS3 (red); EP2AS4 (purple) treated with VlA-specific fluorescent peptide, A647-PhAcALVP.
  • NBS Non-specific binding
  • BMAX Relative cell-surface binding of A647-PhAcALVP
  • VIA mutant single clones determined from saturation binding assays.
  • Each point represents a BMAX value calculated from an individual saturation binding experiment, normalized against the A647-PhAcALVP (BMAX) at cells expressing VIA wt.
  • Columns represent normalized mean BMAX values ⁇ SD.
  • VIA clones 4, 11, 12, 21, 25, 29, 34, 36, 44, and 46 displayed significant increases in expression compared to wild-type upon statistical analysis using one-way ANOVA followed by Dunnett's test.
  • AVP induced Fluo-8 AM calcium mobilization assays in HEK293-T cells transfected with two representative high-expressing VIA mutants compared to VIA wt expressing cells were obtained by subtracting the minimum fluorescence value (before addition of AVP) from maximum fluorescence value (after addition of AVP) for each AVP concentration and normalizing against cells treated with 3 pM ionomycin. Data are mean values from three separate experiments performed in triplicate ⁇ SEM.
  • FIG. 10 shows a single point screen of VIA clones.
  • 23 VIA mutants were isolated from both the EPl AS3 (#1 - 23) and the EP2 AS4 (#24 - 46) population and, along with wild-type VIA (wt - blue), subjected to a single ligand-binding screen.
  • Transfected cells were treated with fluorescent ligand using the same conditions as the sorts (50nM A647-PhAcALVP ⁇ 10 pM SR49059 to determine non-specific binding).
  • Cells were analysed for 655 - 665 nm fluorescence intensity using flow cytometry (A647 MFI), relating to ligand binding signal. Specific binding was determined by subtracting the A647 MFI of the competition sample, obtained with 10 pM SR49059, for each clone.
  • Figure 11 shows the saturation binding curves of VIA clones from bmaxs in
  • Figure 12 shows the saturation binding curves of other VIA clones from bmaxs.
  • Figure 13 shows the ligand binding (A647 fluorescence) of VIA EP2 AS3 cells treated with and without DDM detergent.
  • Figure 14 shows cell-surface expression for selected VIA clones, with the best performing clone #22 exhibiting an approximate 9-fold signal increase over VIA wild-type.
  • Figure 15 shows cell surface expression and detergent stability for clones #6,
  • Figure 16 shows use of a labeled intracellular binding partner to stain and detect membrane targets.
  • the term "about” refers to a quantity, level, value, number, dimension, size, percentage or amount that varies by as much as 10% (e.g., by 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%) to a reference quantity, level, value, number, dimension, size, percentage or amount.
  • encode refers to the capacity of a nucleic acid to provide for another nucleic acid or a polypeptide or protein.
  • a nucleic acid sequence is said to "encode” a polypeptide if it can be transcribed and/or translated to produce the polypeptide or if it can be processed into a form that can be transcribed and/or translated to produce the polypeptide.
  • Such a nucleic acid sequence may include a coding sequence or both a coding sequence and a non-coding sequence.
  • the terms "encode”, "encoding” and the like include an RNA product resulting from transcription of a DNA molecule, a protein resulting from translation of an RNA molecule, a protein resulting from transcription of a DNA molecule to form an RNA product and the subsequent translation of the RNA product, or a protein resulting from transcription of a DNA molecule to provide an RNA product, processing of the RNA product to provide a processed RNA product (e.g., mRNA) and the subsequent translation of the processed RNA product.
  • a processed RNA product e.g., mRNA
  • variants can be used interchangeably herein, to refer to a non-wild-type organism, gene/ polynucleotide sequence nucleic acid, polypeptide or protein, expression pattern or expression level, or amino acid sequence.
  • a variant protein can be a protein that is derived from its corresponding wild-type counterpart.
  • the variant protein can be a protein that is derived from a non-wild-type protein; that is, the variant protein can in turn, be derived from another variant protein.
  • the variant protein may be a naturally occurring variant protein, or may be the product of artificial engineering, selection or mutation.
  • the terms “engineering”, “modification”, “alteration”, “substitution” and the like, as used herein in relation to an amino acid residue/position or a nucleotide typically mean that the amino acid or nucleotide in the particular position has been modified compared to the amino acid of the wild-type or parent polypeptide or protein.
  • the variant will possess at least about 40% identity to the sequence from which it was derived.
  • the variant protein sequence may be at least 50% identical to the sequence from which it was derived.
  • the variant protein sequence may be about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,
  • RNA molecule or a polypeptide typically refers to any step involved in the production of an RNA molecule or a polypeptide, such as by transcription, post- transcriptional modification, translation, post-translational modification, and secretion.
  • isolated is meant material that is substantially or essentially removed from components that normally accompany it in its native state.
  • nucleic acid As used herein, the term "nucleic acid”, “nucleic sequence”, “polynucleotide”,
  • oligonucleotide and “nucleotide sequence” as used herein refers to mRNA, RNA, cRNA, rRNA, cDNA, or DNA, or a combination thereof.
  • the term typically refers to polymeric form of nucleotides, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
  • the term includes single-, double- or triple- stranded forms of DNA and RNA. It can be of recombinant, artificial and /or synthetic origin and it can comprise modified nucleotides, comprising for example a modified bond, a modified purine or pyrimidine base, or a modified sugar.
  • the nucleic acids of the present disclosure can be in isolated or purified form, and made, isolated and /or manipulated by techniques known per se in the art, e.g., cloning and expression of cDNA libraries, amplification, enzymatic synthesis or recombinant technology.
  • the nucleic acids can also be synthesized in vitro by well- known chemical synthesis techniques, as described in, e.g., Belousov et al. (1997).
  • the terms "protein”, “peptide” or “polypeptide” are to be understood as referring to a chain of amino acids linked by peptide bonds, irrespective of the number of amino acids forming said chain.
  • Amino acids are typically represented by their one-letter or three-letters code, according to the following nomenclature: A: alanine (Ala); C: cysteine (Cys); D: aspartic acid (Asp); E: glutamic acid (Glu); F: phenylalanine (Phe); G: glycine (Gly); H: histidine (His); I: isoleucine (lie); K: lysine (Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gin); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Vai); W: tryptophan (Trp) and Y: tyrosine (Tyr).
  • A alanine
  • C cysteine
  • D aspartic acid
  • Glu glutamic acid
  • protein may also be used to refer to such a polymer although in some instances a polypeptide may be shorter (i.e. composed of fewer amino acid residues) than a protein. Nevertheless, the terms “polypeptide” and “protein” may be used interchangeably herein.
  • membrane protein refers to a protein that can be located at the membrane structures of a cell.
  • a membrane protein is a protein that is attached to, interacts with, or associated with the membrane structure.
  • a membrane protein is a protein that forms part of the membrane.
  • the membrane protein can be an integral membrane protein that is permanently attached or associated with the cell membrane.
  • the membrane protein may be a transmembrane protein that spans the entire width of the membrane.
  • the membrane protein may only be attached to one side of the membrane.
  • the membrane protein association with a membrane may be temporary. For example, the membrane protein may be trafficked from one membrane structure to another membrane structure. Alternatively, upon ligand binding or signaling, the membrane protein may dissociate from the membrane.
  • the membrane protein may function to recruit binding of other interacting proteins.
  • the membrane can be the cell membrane or plasma membrane of a cell.
  • the membrane that a membrane protein is associated with can be a membrane of an intracellular organelle.
  • intracellular protein refers to a protein normally residing in the intracellular compartment, i.e. inside the cell.
  • the intracellular protein may be a protein that normally resides in the cytoplasm.
  • the intracellular protein may be a protein that is associated with intracellular structures, wherein the intracellular structures may be a membrane-bound structure I organelle or non-membrane-bound structures.
  • the intracellular protein may be a cytoskeletal protein or a protein associated with the cytoskeleton.
  • the intracellular protein may be a protein associated with the centriole or the microtubule-organizing centre.
  • the term "vector” typically refers to a DNA or RNA molecule used as a vehicle to transfer recombinant genetic material, such as a heterologous nucleic acid construct of the present disclosure, into a host cell.
  • the vector may be a linear or circular double-stranded nucleic acid molecule. Suitable vectors include plasmids, bacteriophages, viruses, fosmids, cosmids, and artificial chromosomes.
  • a vector typically comprises an insert (a heterologous nucleic acid sequence or transgene) and a larger sequence that serves as the "backbone" of the vector.
  • the purpose of a vector which transfers genetic information to the host is typically to isolate, multiply, or express the insert in the target cell.
  • Vectors can be episomal, i.e., do not integrate into the genome of a host cell, or can integrate into the host cell genome.
  • the vectors may also be replication competent or replication-deficient.
  • Exemplary polynucleotide vectors include, but are not limited to, plasmids, yeast artificial chromosomes (YACs), cosmids, transposons, synthetic DNA fragments.
  • Exemplary viral vectors include, for example, AAV, lentiviral, retroviral, adenoviral, herpes viral and hepatitis viral vectors. Selection of the vectors to be used will take into consideration the size of the insert, the host cell to be transfected and the desired transformation efficiency or outcome, and would be readily known to the persons skilled in the art.
  • wild-type is used herein to denote an organism, protein or gene, or gene product, or the expression pattern or expression level of the gene or gene product in a nonmodified organism; that is, as it appears in nature, or that which is most frequently observed in a population and is thus arbitrarily designed the "normal” or "wild-type” form.
  • the present disclosure overcomes a disadvantage identified by the inventors with the selection method described and taught in the prior art in relation to a membrane protein or an intra-cellular protein.
  • the selection method described and taught in the prior art it is preferable to express the proteins in cells and expose them to concentrations of detergent or detergent like compounds that enable selection of the proteins having desired properties.
  • an additional benefit is that the mutation rate in high-expressing GPCR clones appears minimal, preserving the relevance of the clone to the wild-type receptor. This is substantially lower than observed in receptors produced by other directed evolution methods (i.e. in non-mammalian cell systems) which mostly exceed 10 mutations . These minimally mutated clones provided increased yield in purified receptor are more likely to retain normal structure and function. [0085]
  • the development of the mammalian cell-based methods described herein advantageously provide for the purification of substantial amounts of variant proteins having a desired activity, including proteins such as lowly expressed membrane proteins such as receptors. Without wishing to be bound by theory, this method can be applied to other low-expressing, complex membrane proteins or intracellular proteins which are previously difficult to target, and it is anticipated that it can be applied to other protein targets for which similar expression issues have impeded biochemical study.
  • a method for selecting a variant of a protein of interest having a desired activity from a library of expressed nucleic acid sequences encoding variants of the protein of interest comprising: i. providing a population of mammalian cells, wherein each cell of the population of cells comprises and expresses no more than two nucleic acid sequences of said library; ii. exposing the population of cells to a detergent or a detergent-like compound at a concentration sufficient to permeabilise the cell membrane, while retaining the genetic integrity of the exposed cell; ill. contacting the exposed cells with a labelled agent to facilitate detection of activity of the variant proteins encoded by the nucleic acid sequences of said library; and subsequently iv. selecting from the population of cells, a cell expressing a variant protein as a function of the detected agent, wherein the selected cell comprises a variant protein having the desired activity.
  • telomeres In recombinant expression systems (and natural cells) most of the protein pool can be found intracellularly; for example, in the cytoplasm or cytoskeleton, or in intracellular organelles such as the endoplasmic reticulum, Golgi apparatus and the nucleus.
  • a target protein of interest e.g. a GPCR is purified from cells (using high concentrations of detergent), GPCR proteins from all of these intracellular pools are solubilised and purified for downstream use.
  • high recombinant protein expression can only be based solely on the detection of cell-surface localized proteins, and thus may not fully correlate with selection for high purification yields, as it precludes the intracellular pools of protein.
  • the methods described herein advantageously allow selection for cells that express high levels of functional, intracellularly-localized variant proteins, that would greatly benefit purification yields.
  • the methods described herein circumvent issues associated with applying the methods to membrane proteins with extremely low cell-surface expression (such as D2R) and proteins normally residing in the intracellular compartment.
  • a barrier to selection of intracellular protein pools is that most fluorescent ligands, and antibodies, cannot freely enter cells.
  • Previous methods of labelling intracellular proteins or receptors have included the use of fluorescent protein fusion tags (Klenk et al., 2016) which are not true representations of functional receptor as even misfolded proteins will give a fluorescent signal.
  • cell populations are exposed to or treated with detergents such as digitonin (or other saponins), to permeabilise cells, enabling the entry of function-specific labelled agents in order to detect cells that express high levels of proteins of interest
  • detergents such as digitonin (or other saponins)
  • the detergents or detergent-like compounds may function to solubilize cholesterol, leaving most of the cell membrane intact, allowing cells to maintain their overall shape for analysis, such as by flow cytometry and FACS.
  • nucleus of cells treated with such detergents remains intact, allowing downstream isolation of the selected protein of interest or of genetic material pertaining to selected protein of interest (for example in embodiments in which a retrovirus, such as a lentivirus, vector is employed to generate a gene library encoding variant proteins of interest and described herein).
  • a retrovirus such as a lentivirus
  • the desired activity of the selected variant can be an increased expression of the protein of interest, when expressed in a cell.
  • the variant may exhibit an increase in expression by at least about 1%, by at least 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, preferably by at least about 100%, preferably by at least about 200%, preferably by at least about 300%, preferably by at least about 400%, preferably by at least about 500%, preferably by at least about 600%, preferably by at least about 700%, preferably by at least about 800%, preferably by at least about 900%, or more preferably by at least about 1,000% in comparison to the protein from which the variant is derived or the corresponding wild-type protein of interest.
  • the selected variant has the desired activity of increased or decreased affinity for, or binding of, the labelled agent, which would read on the selected variant increased or decreased affinity for, or binding of, a ligand or interacting protein.
  • the variant may exhibit an increase or decrease in affinity for, or binding of, the labelled agent by at least about 1%, by at least 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, preferably by at least about 100%, preferably by at least about 200%, preferably by at least about 300%, preferably by at least about 400%, preferably by at least about 500%, preferably by at least about 600%, preferably by at least about 700%, preferably by at least about 800%, preferably by at least about 900%, or more preferably by at least about 1,000% in comparison to the corresponding wild-type protein of interest.
  • the variant may exhibit an increase or decrease in affinity for, or binding of, a ligand or interacting protein by at least about 1%, by at least 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, preferably by at least about 100%, preferably by at least about 200%, preferably by at least about 300%, preferably by at least about 400%, preferably by at least about 500%, preferably by at least about 600%, preferably by at least about 700%, preferably by at least about 800%, preferably by at least about 900%, or more preferably by at least about 1,000% in comparison to the protein from which the variant is derived or the corresponding wild-type protein of interest.
  • the selected variant has an altered protein-protein interaction with an interacting protein.
  • This altered protein-protein interaction can be a loss of interaction of an interacting protein or it can be an increased interaction with an interacting protein.
  • This altered protein-protein interaction can result in the selected variant having an interaction with a protein that normally does not interact with the corresponding non-variant or wild-type protein of interest.
  • the selected variant has a stabilised conformational state.
  • the protein variant may be stabilised into an active conformational state, an inactive conformational state, an intermediate conformational state, a functionally biased conformational state, or a conformational state where other protein partners a stably bound.
  • a functionally biased conformational state it is meant that the protein variant may favour one or more of a plurality of functions or activities.
  • a functionally biased receptor variant may preferentially activate one pathway over another, optionally independent of the agonist present, and a functionally biased agonist may preferentially activate one pathway over another.
  • the selected variant has a modified protein trafficking to the cell membrane or modified protein trafficking to the cell nucleus or modified protein trafficking to the mitochondria, when compared to the protein from which the variant is derived or the corresponding wild-type protein of interest.
  • the selected variant may be selected for altered protein halflife (i.e. increased protein half-life or decreased protein half-life), improved or altered solubility, pH stability, thermostability, detergent stability, folding properties, binding characteristics, improved performance and/or novel functionalities.
  • the population of cells are exposed to a detergent or a detergent-like compound at a concentration sufficient to permeabilise the cell membrane, while retaining the genetic integrity of the exposed cell.
  • the population of cells that are exposed to a detergent or a detergent-like compound are live cells.
  • the population of cells that are exposed to a detergent or a detergent-like compound are cells that have been fixed or partially fixed.
  • the population of cells are fixed or partially fixed before exposure to a detergent or a detergent-like compound. Partial fixation or fixation can be used to preserve or stabilize cell architecture or prevent complete loss of soluble proteins.
  • the cells can be fixed using preservatives, selection of which would be known to the skilled person. Examples of suitable cell fixatives or preservation agents include methanol, ethanol, acetone, formaldehyde, paraformaldehyde, formalin or glutaraldehyde.
  • the detergent or detergent-like compound may be a saponin.
  • the detergent or detergent-like compound is digitonin, diosgenin, glycol-diosgenin (GDN), or other saponins.
  • the detergent or detergent-like compound is digitonin. Permeabilisation of cell membrane enables the entry of labelled agents such an exogenous sensor/ligand/antibody for selection, through the cell membrane and into the intracellular compartment of the cell to access proteins that are otherwise cell-impermeable.
  • Permeabilisation of the cell membrane as described herein refers to the formation of cell membrane pores, or the puncturing of holes in the cell membrane, while retaining the genetic integrity of the exposed cells.
  • the permeabilisation of the cell membrane of the exposed cell must not disrupt or compromise the genetic material of the cell such that the genetic material of the cell cannot be isolated or purified.
  • the cell membrane of the exposed cell is permeabilised without destroying the cell membrane.
  • the cell membrane of the exposed cell is permeabilised while retaining cell structure.
  • the cell membrane of the exposed cell is permeabilised while retaining cell viability. Retaining the viability of the permeabilised cell, means the cell can be recovered and further cultured. After selection, the selected cell can expanded for cell number and/or subject to further rounds of exposure/permeabilisation, contacting and selection.
  • the cells may be recovered for further cell culture or passaging.
  • the selected cell may be one or more cells expressing a variant membrane protein.
  • single selected cells expressing a variant membrane protein of interest can be isolated and cultured for clonal expansion.
  • a plurality of selected cells can be pooled to give generate a population of cells enriched for cells expressing variant proteins, and be subjected to further rounds of detergent exposure, contacting with a labelled agent and selection.
  • the methods disclosed herein further comprise purifying the variant protein of interest from the selected cell.
  • the methods disclosed herein further comprise a) isolating from the selected cell, the nucleic acid sequence encoding the variant protein having the desired activity; b) introducing, transfecting or transducing the nucleic acid sequence encoding the variant protein having the desired activity into a population of mammalian cells, to generate a population of cells expression variant proteins having the desired activity, and repeating the steps of detergent exposure, contacting with a labelled agent and selection.
  • the methods disclosed herein further comprise isolating from the selected cell, the nucleic acid sequence encoding the variant protein having the desired activity; b) generating one or more mutations in the isolated sequence to produce a second library of nucleic acid sequences encoding variant proteins having the desired activity; c) introducing said library of nucleic acid sequences encoding variant proteins having the desired activity into a second population of cells to provide a population of mammalian cells, wherein each cell of the population of cells comprises and expresses no more than two nucleic acid sequences of said second library, and repeating the steps of detergent exposure, contacting with a labelled agent and selection.
  • the protein of interest is a membrane protein.
  • the protein may be an outer mitochondrial membrane protein, a resident membrane protein of the endoplasmic reticulum or Golgi apparatus, a scaffolding protein, or a nuclear protein such as a nuclear pore complex member.
  • the protein of interest is an intracellular protein.
  • the protein of interest is a protein that can be found in or is associated with intracellular organelles, such as the endoplasmic reticulum, Golgi apparatus, the cytoskeleton, or the nucleus.
  • the protein of interest is a protein that can be found in or is associated with intracellular membranes.
  • the protein of interest is a receptor protein, an ion channel, a tyrosine kinase or a transporter protein.
  • the protein of interest is a G-protein coupled receptor (GPCR).
  • GPCR G-protein coupled receptor
  • the protein of interest is the dopamine receptor D2.
  • the protein of interest is the serotonin receptor 5-HT2A.
  • the protein of interest is the neurotensin receptor (NTR).
  • the protein of interest is a chimeric protein.
  • a chimeric protein may also be referred to as a fusion protein, that is a protein that is created through the joining of two or more peptide or polypeptide sequences from different proteins, polypeptides or peptides.
  • Such chimeric molecules may be produced by recombinant DNA technology using methods well known to those skilled in the art.
  • the chimeric protein may be a protein that arises from the fusion of domains that originate from different proteins that may be related.
  • the chimeric protein may be a protein that arises from the fusion of domains that originate from different proteins that may be not related to each other.
  • the chimeric protein may comprise one or more domains from one or more different proteins, while retaining function and/or activity in common with one or more of the proteins from which the domains are derived.
  • the chimeric protein may comprise one or more domains from one or more different proteins, and have new or different function and/or activity when compared to the proteins from which the domains are derived.
  • the chimeric protein may be a chimeric receptor, such as a chimeric GPCR. Examples of chimeric receptors and chimeric GPCRs, methods of their generation and characterization are described in Gearing et al., 2003; Hani et al., 2002; Jewell-Motz et al.
  • the chimeric GPCR may comprise one or more domains, for example transmembrane domains, from different GPCR proteins.
  • the chimeric GPCR may comprise one or more transmembrane domains of the kappa opioid receptor.
  • the chimeric GPCR may comprise an intracellular loop of the kappa opioid receptor.
  • the chimeric GPCR may comprise a nanobody binding epitope.
  • the present disclosure provides a method for selecting a variant of a chimeric GPCR of interest from a library of expressed nucleic acid sequences encoding variant chimeric GPCRs, wherein the chimeric GPCR comprises an Nb6 binding epitope, the method comprising:
  • each cell of the population of cells comprises and expresses no more than two nucleic acid sequences of said library; ii. exposing the population of cells to a detergent or a detergent-like compound at a concentration sufficient to permeabilise the cell membrane, while retaining the genetic integrity of the exposed cell; ill. contacting the exposed cells with a labelled Nb6 to facilitate detection of a conformational state of the chimeric GPCR encoded by the nucleic acid sequences of said library; and subsequently iv.
  • the present disclosure provides a method for selecting a variant of a chimeric GPCR of interest from a library of expressed nucleic acid sequences encoding variant chimeric GPCRs, wherein the chimeric GPCR comprises a G protein binding site or a mini G protein binding site, the method comprising: i. providing a population of mammalian cells, wherein each cell of the population of cells comprises and expresses no more than two nucleic acid sequences of said library; ii.
  • the detergent or detergent-like compound is digitonin or
  • the chimeric GPCR may be a functional GPCR. In another embodiment the chimeric GPCR may be a detergent-stable chimeric GPCR.
  • the chimeric GPCR may comprise domains of the kappa opioid receptor. In one embodiment, the chimeric GPCR may comprise the transmembrane domains of the kappa opioid receptor In one embodiment, the chimeric GPCR may comprise the intracellular loops of the kappa opioid receptor. Some examples of chimeric GPCRs and their generation are provided in Che et al., 2020. In another example, the chimeric GPCR comprises the intracellular ends of transmembrane 5 and transmembrane 6. In another example, the chimeric GPCR comprises the intracellular ends of transmembrane 5 and transmembrane 6 and/or the intracellular loop 3 domain from the kappa opioid receptor.
  • the chimeric receptor comprises an Nb6 binding epitope (Che et al., 2020; Robertson et al., 2022).
  • the Nb6 binding epitope comprises the intracellular ends of transmembrane 5, the intracellular ends of transmembrane 6, and/or the intracellular loop 3 domain from the kappa opioid receptor (KOR).
  • the Nb6 binding epitope comprises the amino acid sequence of any one of SEQ ID NOs: 15-21.
  • the Nb6 binding epitope may comprise the amino acid of SEQ ID NO: 16.
  • the Nb6 binding epitope may comprise the amino acid of SEQ ID NO: 17.
  • the Nb6 binding epitope may comprise the amino acid of SEQ ID NO: 18.
  • the Nb6 binding epitope may comprise the amino acid of SEQ ID NO: 19.
  • the Nb6 binding epitope may comprise the amino acid of SEQ ID NO:20.
  • the Nb6 binding epitope may comprise the amino acid of SEQ ID NO:21.
  • the Nb6 binding epitope comprises the amino acid sequence of SEQ ID NO: 15.
  • Also provided herein is a method for selecting a detergent-stable functional variant of a membrane protein of interest from a library of expressed nucleic acid sequences encoding variant membrane proteins, the method comprising: i. providing a population of mammalian cells, wherein each cell of the population of cells comprises and expresses no more than two nucleic acid sequences of said library;
  • sub-CSC sub-critical solubilisation concentration
  • detergent-stable refers to the characteristic ability of a protein to maintain its structure, conformation or function while resisting aggregating, precipitating, unfolding or denature when exposed to detergents.
  • the term can be taken to refer to a characteristic of a protein having reduced detergent sensitivity. That is, a detergent-stable protein or a protein having reduced detergent sensitivity is able to maintain its structure, conformation or function while resisting aggregating, precipitating, unfolding or denature when exposed to detergents.
  • a detergent-stable protein is one that maintain its structure, conformation or function while resisting aggregating, precipitating, unfolding or denature when exposed to a sub-critical solubilisation concentration (sub-CSC) of detergent or a detergent like compound.
  • sub-CSC concentration of detergent or a detergent like compounds will be known or determined by the person skilled in the art, for example, using the methods described herein.
  • the selected detergent-stable variant membrane protein can have increased detergent stability or reduced detergent sensitivity, of at least about 1%, by at least 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, preferably at least about 50%, preferably by at least about 100%, preferably by at least about 200%, preferably by at least about 300%, preferably by at least about 400%, preferably by at least about 500%, in comparison to the protein from which the variant is derived or the corresponding wild-type protein of interest.
  • Methods of measuring detergent stability of proteins and membrane proteins are described herein. Alternative methods of measuring detergent stability of proteins and membrane proteins are well-known in the art, for example as described in Scott & Pluckthun (2013), Tomasiak et al. (2014), Wang et al. (2015), Hattori et al. (2012), Sonoda et al. (2011).
  • the term "functional" when used in relation to the detergentstable variant membrane protein refers to the variant protein having a qualitative activity in common with the corresponding wild-type membrane protein or the membrane protein from which it is derived.
  • the functional variant membrane protein may retain ligand binding activity, signaling capacity, activation capacity, ability to interact with other proteins and/or retain its structure.
  • the functional variant membrane protein may demonstrate increased or reduced activity or function relative to the activity of wild-type protein or the membrane protein from which it is derived.
  • the selected detergent-stable functional variant membrane protein can have increased activity or function of at least about 1%, by at least 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, preferably at least about 50%, preferably by at least about 100%, preferably by at least about 200%, preferably by at least about 300%, preferably by at least about 400%, preferably by at least about 500%, in comparison to the corresponding wild-type protein of interest.
  • the selected detergent-stable functional variant membrane protein can have reduced activity or function of at least about 1%, by at least 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, preferably at least about 50%, preferably by at least about 100%, preferably by at least about 200%, preferably by at least about 300%, preferably by at least about 400%, preferably by at least about 500%, in comparison to the protein from which the variant is derived or the corresponding wild-type protein of interest.
  • the detergent-stable variant membrane protein may possess one or more desired activities or properties.
  • the selected variant may be selected for altered protein halflife (i.e. increased protein half-life or decreased protein half-life), improved or altered solubility, pH stability, thermostability, detergent stability, folding properties, binding characteristics, improved performance and/or novel functionalities.
  • the desired activity of the selected variant can be an increased expression of the protein of interest, when expressed in a cell.
  • the variant may exhibit an increase in expression by at least about 1%, by at least 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, preferably by at least about 100%, preferably by at least about 200%, preferably by at least about 300%, preferably by at least about 400%, preferably by at least about 500%, preferably by at least about 600%, preferably by at least about 700%, preferably by at least about 800%, preferably by at least about 900%, or more preferably by at least about 1,000% in comparison to the protein from which the variant is derived or the corresponding wild-type protein of interest.
  • the desired activity may comprise increased or decreased affinity for, or binding of, the labelled agent, which would read on the selected variant increased or decreased affinity for, or binding of, a ligand or interacting protein.
  • the variant may exhibit an increase or decrease in affinity for, or binding of, the labelled agent by at least about 1%, by at least 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, preferably by at least about 100%, preferably by at least about 200%, preferably by at least about 300%, preferably by at least about 400%, preferably by at least about 500%, preferably by at least about 600%, preferably by at least about 700%, preferably by at least about 800%, preferably by at least about 900%, or more preferably by at least about 1,000% in comparison to the corresponding wildtype protein of interest.
  • the variant may exhibit an increase or decrease in affinity for, or binding of, a ligand or interacting protein by at least about 1%, by at least 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, preferably by at least about 100%, preferably by at least about 200%, preferably by at least about 300%, preferably by at least about 400%, preferably by at least about 500%, preferably by at least about 600%, preferably by at least about 700%, preferably by at least about 800%, preferably by at least about 900%, or more preferably by at least about 1,000% in comparison to the protein from which the variant is derived or the corresponding wild-type protein of interest.
  • the selected variant may have an altered protein-protein interaction with an interacting protein.
  • This altered protein-protein interaction can be a loss of interaction of an interacting protein or it can be an increased interaction with an interacting protein.
  • This altered protein-protein interaction can result in the selected variant having an interaction with a protein that normally does not interact with the corresponding non-variant or wild-type protein of interest.
  • the selected variant may have a stabilised conformational state.
  • the protein variant may be stabilised into an active conformational state, an inactive conformational state, an intermediate conformational state, a functionally biased conformational state, or a conformational state where other protein partners a stably bound.
  • a functionally biased conformational state it is meant that the protein variant may favour one or more of a plurality of functions or activities.
  • a functionally biased receptor variant may preferentially activate one pathway over another, optionally independent of the agonist present, and a functionally biased agonist may preferentially activate one pathway over another.
  • the selected variant may have a modified protein trafficking to the cell membrane or modified protein trafficking to the cell nucleus or modified protein trafficking to the mitochondria, when compared to the protein from which the variant is derived or the corresponding wild-type protein of interest.
  • the method may further comprise recovering the selected cells and repeating the steps of exposing the population of cells to a sub-critical solubilisation concentration (sub-CSC) of a detergent or a detergent-like compound, contacting the exposed cells with a labelled agent to facilitate detection of an activity of the variant membrane protein, and selecting from the population of cells, a cell expressing a variant membrane protein as a function of the detected agent, wherein the selected cell comprises a detergent-stable functional variant membrane protein.
  • the step of recovering the selected cells can include culturing and/or expansion of the selected cells.
  • the recovery of the selected cells can include clonal expansion of the selected cell, or expansion of a pool of selected cells.
  • Suitable cells include, but are not limited to, bacterial cells, yeast cells, protozoal cells, algal or other plant cells, or an animal cells, such as insect or mammalian cells.
  • the cell may be a primary or secondary cell culture or an immortalized cell line.
  • the cell is a mammalian cell or cell line, optionally a human cell or cell line.
  • the human cell or cell line may be, for example, an embryonic or stem cell or cell line.
  • any cell may be employed, and scope of the present disclosure is not limited by the identity or origin of the cell selected for any particular application.
  • the selected variant has increased expression when expressed in a cell.
  • the variant may exhibit an increase in expression by at least about 1%, by at least 5%, preferably by at least about 10%, preferably by at least about 20%, preferably by at least about 30%, preferably by at least about 40%, preferably by at least about 50%, preferably by at least about 100%, preferably by at least about 200%, preferably by at least about 300%, preferably by at least about 400%, preferably by at least about 500%, preferably by at least about 600%, preferably by at least about 700%, preferably by at least about 800%, preferably by at least about 900%, or more preferably by at least about 1,000% in comparison to the protein from which the variant is derived or the corresponding wild-type protein of interest.
  • the population of cells are exposed to a sub-critical solubilisation concentration (sub-CSC) of a detergent or a detergent-like compound.
  • sub-CSC sub-critical solubilisation concentration
  • the population of cells that are exposed to a detergent or a detergent-like compound are live cells.
  • the population of cells that are exposed to a detergent or a detergent-like compound are cells that have been fixed or partially fixed.
  • the population of cells are fixed or partially fixed before exposure to a detergent or a detergent-like compound.
  • Partial fixation or fixation can be used to preserve or stabilize cell architecture or prevent complete loss of soluble proteins.
  • the cells can be fixed using preservatives, selection of which would be known to the skilled person. Examples of suitable cell fixatives or preservation agents include methanol, ethanol, acetone, formaldehyde, paraformaldehyde, formalin or glutaraldehyde.
  • Table 1 provides a list of detergents or detergent-like compounds that can be used in the methods of the present invention.
  • the detergent or detergentlike compound is a neopentyl glycol (NG) detergent or a maltoside detergent.
  • the detergent or detergent-like compound is an alkyl maltoside.
  • the detergent or detergent-like compound is LMNG.
  • the detergent or detergent-like compound is DDM.
  • Critical solubilisation concentration is defined as is the amount of a detergent or a detergent-like compound required to disrupt a membrane system into a predominantly micellar dispersion. When a cell is in the presence of the CSC of detergent, it will disintegrate the lipid bilayer, solubilise membrane proteins, and the contents of the membrane are incorporated into detergent micelles, resulting in irreversible solubilisation of the cells.
  • the CSC is an intrinsic property of the detergent selected, and can depend on solubilisation conditions, which will be known to the person skilled in the art.
  • sub-CSC detergent or a detergent-like compound at a level below CSC
  • the detergent molecules gradually interact and partition or integrate into cell membranes, but there is no formation of a micelle.
  • the membrane system may be altered by incorporation of the detergent, but remains intact.
  • the alteration of the membrane structure may be reversible, and the integrity of the cell membrane and the internal contents of the cell is retained. This beneficially maintains the viability and genomic integrity of the cell, and allows further culture and expansion of the treated cells.
  • sub-CSC is a concentration of a detergent or a detergent-like compound at a level below the CSC of the detergent or the detergent-like compound.
  • a cell expressing a detergent-stable functional variant of a membrane protein of interest as a function of the detected agent, wherein the selected cell comprises the detergent-stable functional variant membrane protein the cells may be recovered for further cell culture or passaging.
  • the selected cell may be one or more cells expressing a detergent-stable functional variant membrane protein.
  • single selected cells expressing a detergent-stable functional variant membrane protein can be isolated and cultured for clonal expansion.
  • a plurality of selected cells can be pooled to give generate a population of cells enriched for cells expressing detergent-stable functional variant membrane proteins, and be subjected to further rounds of detergent exposure, contacting with a labelled agent and selection.
  • the methods disclosed herein further comprise purifying the detergent-stable functional variant membrane protein from the selected cell.
  • the methods disclosed herein further comprise a) isolating from the selected cell, the nucleic acid sequence encoding the detergent-stable functional variant membrane protein; b) introducing, transfecting or transducing the nucleic acid sequence encoding the detergent-stable functional variant membrane protein into a population of mammalian cells, to generate a population of cells expression detergent-stable functional variant membrane protein, and repeating the steps of detergent exposure, contacting with a labelled agent and selection.
  • the methods disclosed herein further comprise isolating from the selected cell, the nucleic acid sequence encoding the detergent-stable functional variant membrane protein; b) generating one or more mutations in the isolated sequence to produce a second library of nucleic acid sequences encoding detergent-stable functional variant membrane protein; c) introducing said library of nucleic acid sequences encoding detergent-stable functional variant membrane protein into a second population of cells to provide a population of mammalian cells, wherein each cell of the population of cells comprises and expresses no more than two nucleic acid sequences of said second library, and repeating the steps of detergent exposure, contacting with a labelled agent and selection.
  • the membrane protein of interest is an integral membrane protein.
  • the membrane protein is a cell adhesion molecule.
  • the membrane protein is a membrane enzyme.
  • the membrane protein of interest is a receptor protein, an ion channel, a tyrosine kinase or a transporter protein.
  • the membrane protein is a G-protein coupled receptor (GPCR).
  • GPCR G-protein coupled receptor
  • the membrane protein is the vasopressin receptor, VIA.
  • the membrane protein is the dopamine receptor D2.
  • the membrane protein is the serotonin receptor 5-HT2A.
  • the membrane protein is the neurotensin receptor (NTR).
  • the membrane protein is a chimeric protein.
  • a chimeric protein may also be referred to as a fusion protein, that is a protein that is created through the joining of two or more peptide or polypeptide sequences from different proteins, polypeptides or peptides. Such chimeric molecules may be produced by recombinant DNA technology using methods well known to those skilled in the art.
  • the chimeric membrane protein may be a protein that arises from the fusion of domains that originate from different proteins that may be related.
  • the chimeric protein may be a protein that arises from the fusion of domains that originate from different proteins that may be not related to each other.
  • the chimeric protein may comprise one or more domains from one or more different proteins, while retaining function and/or activity in common with one or more of the proteins from which the domains are derived.
  • the chimeric protein may comprise one or more domains from one or more different proteins, and have new or different function and/or activity when compared to the proteins from which the domains are derived.
  • the chimeric protein may be a chimeric receptor, such as a chimeric GPCR.
  • the chimeric GPCR may comprise one or more domains, for example transmembrane domains, from different GPCR proteins.
  • the chimeric receptor may comprise a G protein binding site or a mini G protein binding site.
  • the chimeric receptor may comprise a nanobody binding epitope.
  • the chimeric GPCR may comprise one or more transmembrane domains of the kappa opioid receptor.
  • the chimeric GPCR may comprise the intracellular loops of the kappa opioid receptor.
  • the chimeric GPCR comprises the intracellular ends of transmembrane 5 and transmembrane 6, and/or the intracellular loop 3 domain from the kappa opioid receptor.
  • the chimeric receptor comprises an Nb6 binding epitope (Che et al., 2020).
  • the Nb6 binding epitope comprises the intracellular ends of transmembrane 5, the intracellular ends of transmembrane 6, and/or the intracellular loop 3 domain from the kappa opioid receptor (KOR).
  • the Nb6 binding epitope comprises the amino acid sequence of any one of SEQ ID NOs: 15-21.
  • the Nb6 binding epitope may comprise the amino acid of SEQ ID NO: 16.
  • the Nb6 binding epitope may comprise the amino acid of SEQ ID NO: 17.
  • the Nb6 binding epitope may comprise the amino acid of SEQ ID NO: 18.
  • the Nb6 binding epitope may comprise the amino acid of SEQ ID NO: 19.
  • the Nb6 binding epitope may comprise the amino acid of SEQ ID NO:20.
  • the Nb6 binding epitope may comprise the amino acid of SEQ ID NO:21.
  • the Nb6 binding epitope comprises the amino acid sequence of SEQ ID NO: 15. Detergents and detergent-like compounds
  • the methods of selecting a variant of a protein of interest and methods of selecting a detergent-stable functional variant membrane protein involve a step of exposing the cells expressing the variant proteins or detergent-stable functional variant membrane protein to detergents or detergent like compounds.
  • Non-limiting examples of detergents or detergent-like compounds is shown in Table 1.
  • the population of cells expressing variant proteins are exposed to a detergent or a detergent-like compound at a concentration sufficient to permeabilise the cell membrane, while retaining the genetic integrity of the exposed cell.
  • the detergent or detergent-like compound may be a saponin.
  • the detergent or detergent-like compound is digitonin, diosgenin, glycol- diosgenin (GDN), or other saponins.
  • the detergent or detergentlike compound is digitonin.
  • the concentration of digitonin, diosgenin and other saponins useful to permeabilise the cell membrane while retaining the genetic integrity of the exposed cell can range from 0.0001 - 0.01 % (w/v) (approximately 1 - 100 pM) and may vary depending on, for example, the specific cell type. For example, cells (especially non-mammalian cells) with low or no cholesterol-containing cell membranes may require concentrations of digitonin, diosgenin and other saponins that are at the higher end of this range in order to permeabilise the cell membrane. By way of example, concentrations below this range may not sufficiently permeabilise the cell membrane. Concentrations above this range risk irreversible disruption of the cell membrane, fully solubilizing cells and leading to loss of cell integrity and genetic integrity.
  • the population of cells expressing detergent-stable functional variant membrane proteins are exposed to a sub-critical solubilisation concentration (sub-CSC) of a detergent or a detergent-like compound.
  • the CSC of a detergent is the amount required to disrupt a membrane system into a predominantly micellar dispersion (Chattopadhyay et al., 2015; Prive, 2007). (Chattopadhyay et al., 2015; Prive, 2007).
  • the CSC is an intrinsic property of the detergent selected, as well as solubilization conditions, including cell membrane concentration, temperature and time, which would be known to persons skilled in the art.
  • the detergent or detergent-like compound is a neopentyl glycol (NG) detergent or a maltoside detergent.
  • the detergent or detergent-like compound is an alkyl maltoside.
  • the detergent or detergent-like compound is LMNG.
  • the detergent or detergentlike compound is DDM.
  • a concentration of detergent and incubation conditions that would leave a membrane system intact, to maintaining the cell content and the retain genetic information inside the cell nucleus (thus maintaining genetic integrity).
  • it is desirable to maintain cell viability throughout the selection process enables the cells to be cultured straight after rounds of selection, making iterative selections more efficient.
  • cells may be exposed to a sub-critical solubilisation concentration (sub-CSC) of a detergent or a detergent-like compound.
  • sub-CSC sub-critical solubilisation concentration
  • the exposed cells are contacted with a labelled agent to facilitate detection of an activity of the variant membrane protein or the variant membrane protein; and subsequently selecting from the population of cells, a cell expressing a variant membrane protein or a variant membrane protein as a function of the detected agent.
  • the agent may comprise, for example, ligands, proteins, antibodies, antibody fragment, nanobodies, substrates or other biosensors capable of facilitating detection of protein activity or function, such as those herein below described. Agents facilitate detection of an activity of the variant membrane protein or variant membrane protein of interest and that are suitable for use in the methods disclosed herein can be identified by those of skill in the art.
  • the agent contains a label.
  • the agent is labelled with a fluorescent dye or a fluorescent label (e.g.
  • the label is an isotope, a magnetic particle or a dye. It is important that the label is a detectable label.
  • the agent can be a ligand wherein the ligand can be a receptor ligand, wherein the protein of interest is a receptor.
  • the ligand is an enzyme substrate, where binding of the enzyme protein variant (which is an enzyme in this embodiment) results in the generation of a detectable signal.
  • Ligands may be nucleic acid molecules, peptides or proteins including, for example, natural ligands of the protein, as well as engineered protein ligands such as antibodies and fragments thereof (e.g. Fab fragment, scFv, sdAb (i.e. nanobodies)).
  • selection of protein variants would be based on increased detection of binding of a labelled agent, wherein the labelled agent comprises an agent that binds the protein of interest.
  • selection of protein variants may be based on increased capacity (e.g. affinity or amount) to bind to a conformational stabilizing labelled agent (such as an antibody, a nanobody, G proteins or mini G protein probes).
  • Mini G proteins are engineered GTPase domains of Go subunits that were developed for structural studies of GPCR conformation states, and are useful surrogates for the study of GPCR activation in cells.
  • mG proteins have been described as having maintain the receptor-coupling specificity of the different Go subunit families (see for example Wan et al., 2018; Nehme et al., 2017).
  • Other examples of labelled protein agents e.g. labelled engineered arrestins and opsins
  • capable of facilitating detection of protein activity or function of the variant membrane protein or variant membrane protein of interest and that are suitable for use in the methods disclosed herein include those described in Lohse et al., 2012 and Ballister et al., 2018.
  • nanobody refers to a single domain antigen binding fragment, optionally to a single variable domain derived from a naturally occurring heavy chain antibody. Nanobodies are usually derived from heavy chain only antibodies (devoid of light chains) seen in camelids and consequently are often referred to as VHH antibody or VHH sequence. As single-domain antibodies developed from camelids, nanobodies display unique properties such as small-size and high-affinity. It should be understood that nanobodies should not be limited to a specific biological source or to specific methods of preparation, which would be known to the person skilled in the art.
  • Nanobodies have been used to recognize and/or stabilize specific receptor states, including to stabilize the active states of many GPCRs, including the 82- adrenergic ( 2AR), M2-muscarinic (M2R), p-opioid (MOR), k-opioid (KOR), and angiotensin II type 1 (AT1R) receptors. Nanobodies also act as allosteric effectors and can affect both ligand binding affinity and functional activity, such as stabilization of an active or inactive conformational state.
  • 2AR 82- adrenergic
  • M2R M2-muscarinic
  • MOR p-opioid
  • KOR k-opioid
  • A1R angiotensin II type 1
  • Nanobodies can therefore be used as conformational biosensors to monitor GPCR dynamics both in vitro and in vivo, examples of which have been disclosed in WO2020221768, W02015121092 and W02012007594.
  • the nanobody is capable of detecting the inactive state of a GPCR, for example those described in Robertson et al. 2022.
  • a nanobody that may be used in the methods disclosed herein is a negative allosteric nanobody that binds the kappa opioid receptor and stabilizes a ligand-dependent inactive state, such as Nb6 (Che et al., 2018 and Che, et al., 2020).
  • a nanobody that may be used in the methods disclosed herein may be used for detecting the active state of a GPCR, for example Nb80 described in Rasmussen et al., 2011.
  • the nanobody that may be used in the methods disclosed herein is a positive allosteric nanobody that binds the kappa opioid receptor and stabilizes a fully active state, such as Nb39 (Che et al., 2018).
  • nanobody 6 that may be used in the methods disclosed herein is nanobody 6 (Nb6) that recognizes the inactive state conformation (stabilised by receptor inverse agonist binding and de-stabilised by receptor agonist binding) of the extracellular ends of transmembrane 5 8i 6 of the KOR (Che et al., 2020).
  • the selection step comprises cell sorting.
  • the flow cytometry is fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • the selection step comprises a single round of cell sorting.
  • the selection step comprises more than one consecutive round of cell sorting.
  • the selection step comprises 2- 20 consecutive rounds of cell sorting.
  • the selection step comprises 2-15 consecutive rounds of cell sorting.
  • the selection step comprises 2-10 consecutive rounds of cell sorting.
  • the selection step comprises 2-5 rounds of cell sorting.
  • selection of functional protein variants and mutants may be based on fluorescence readouts of protein function such as dimerization of the protein, interaction with other cellular proteins, stimulation of cell-signaling pathways, activation of gene transcription, kinase activation, activation of ion channels, activation of protein degradation, internalization of proteins, membrane reorganization, activation of cellular enzymes, and activation of protein trafficking.
  • Such fluorescence readouts may be obtained by, for example, staining cells with fluorescent dyes or reporter dyes, recombinant expression of fluorescent protein-fused proteins, recombinant expression of bimolecular-fluorescent complementation partner fused proteins, recombinant expression of fluorescent protein-fused proteins where the fluorescent proteins are pairs for fluorescent-resonance energy transfer (FRET) detection of protein-protein interactions, recombinant expression of signaling sensors (e.g. CAMYEL FRET sensor for cAMP, GCaMP for calcium, voltage sensors), or reporter genes expressing fluorescent proteins or enzymes.
  • FRET fluorescent-resonance energy transfer
  • FRET donor/acceptor fluorescent protein-GPCR fusion protein may be co-expressed with FRET donor/acceptor fluorescent protein- G proteins or arrestin protein and interactions between the GPCR and these effector proteins monitored using FRET or monitoring receptor dimerization (see, for example, Vietnameser and Eidne, 2005.
  • FRET-based sensors bioluminescence resonance energy transfer (BRET)-based sensors and lanthanide-based homogeneous time resolved fluorescence (HTRF) sensors.
  • suitable sensors are described, for example, in Tainaka et al., 2010.
  • cells may be labelled with a calcium-sensing dye and receptor- induced calcium signaling monitored, receptor activation of specific genes may be monitored using reporter assays (see, for example, Hill et al., 2001), or a FRET based signaling sensor such as CAMYEL may be co-expressed (see, for example, Matthiesen and Nielsen, 2011).
  • reporter assays see, for example, Hill et al., 2001
  • FRET based signaling sensor such as CAMYEL may be co-expressed (see, for example, Matthiesen and Nielsen, 2011).
  • nucleic acid sequences encoding variants of a protein can be prepared and transfected or transduced into cells using any method known to those skilled in the art.
  • the nucleic acid sequences encoding variants of a protein can be mRNA, RNA, cRNA, rRNA, cDNA, or DNA, or a combination thereof.
  • Such a nucleic acid sequence may include a coding sequence.
  • Such a nucleic acid sequence may also include a non-coding sequence, examples of which include regulatory elements such as promoters, introns, and upstream untranslated regions and downstream untranslated regions (5' and 3' UTRs).
  • Suitable vectors for use in transducing cells in accordance with the present disclosure include retrovirus vectors, adenovirus vectors and adeno- associated virus vectors.
  • the vector is a retrovirus vector.
  • a suitable retrovirus-based system comprises lentiviral vectors and transduction.
  • a number of lentiviral vector and transduction systems suitable for use in accordance with the present disclosure are commercially available and are well known to those skilled in the art.
  • the library of nucleic acid sequences is introduced into the population of cells using a lentiviral vector.
  • the lentiviral vector comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 1.
  • the lentiviral vector comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 1.
  • the lentiviral vector comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 1.
  • the lentiviral vector comprises a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 1.
  • the lentiviral vector comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 1.
  • the lentiviral vector comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1. In one embodiment, the lentiviral vector comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO: 1. In one embodiment, the lentiviral vector comprises a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 1. In a preferred embodiment, the lentiviral vector comprises a nucleic acid sequence of SEQ ID NO: 1.
  • the lentiviral vector comprises one or more of: a) HIV-1 5' and 3' long terminal repeats; b) a psi packaging signal; c) a cloning site; d) a Cytomegalovirus promoter; e) an IRES; and f) one or more selectable markers.
  • the selectable marker is a fluorescent protein gene, molecular marker gene, an antibiotic resistance gene or SacB gene.
  • small-molecular weight proteins are the protein of interest, they can be produced as fusions to other oligopeptides or proteins to form larger structures (e.g. a triple GFP tag).
  • gene libraries can in some embodiments include fusion genes that encode fusion proteins.
  • Gene diversification or gene mutations can involve random mutagenesis, focused mutagenesis or a combination thereof. These methods include, but are not limited to, chemical or environmental mutagenesis (e.g. nitrous acid, UV irradiation and bisulfite), error prone PCR, site directed saturation mutagenesis, homologous recombination (e.g.
  • DNA shuffling DNA shuffling, family shuffling, staggered extension process (StEP), random chimeragenesis on transient templates (RACHITT), nucleotide exchange and excision technology (NExT), heritable recombination, assembly of designed oligonucleotides (ADO) and synthetic shuffling) and non-homologous recombination (e.g. incremental truncation for the creation of hybrid enzymes (ITCHY), sequence homology-independent protein recombination (SHIPREC), non- homologous random recombination (NRR), sequence-independent site-directed chimeragenesis (SISDC) and overlap extension PC) (see, reviewed in Packer and Liu, 2015). Mutations can also be introduced into a nucleic acid sequence by rational design of mutations and generated synthetically.
  • StEP staggered extension process
  • RACHITT random chimeragenesis on transient templates
  • NxT nucleotide exchange and excision technology
  • ADO assembly of
  • the library of nucleic acid sequences is introduced into the population of cells to achieve a multiplicity of infection (MOI) that minimizes double-transductions.
  • MOI multiplicity of infection
  • the library of nucleic acid sequences is introduced into the population of cells in such a way that each cell of the population of cells comprises and expresses no more than two nucleic acid sequences of said library.
  • the library of nucleic acid sequences is introduced into the population of cells in such a way that each cell of the population of cells comprises and expresses no more than one nucleic acid sequence of said library.
  • MOI is an estimation of the ratio of viral particles to target cells, or the average number of viral particles to infect any given cell.
  • the MOI provides a guide to titrating a viral transduction, where the number of virions infecting a target cell is given as a normal distribution, allowing the estimation of infection probabilities.
  • m represents the MOI
  • n represents the number of virions to infect a target cell.
  • Error prone PCR may be used to generate a diverse library of the target gene.
  • the library can then be ligated into a customized lentiviral transfer plasmid.
  • Cells can be transfected with lentiviral components, for example customized transfer plasmid, packaging plasmids, and envelope plasmid, and lentivirus-containing media then harvested.
  • Cells can be transduced with library-containing lentivirus at a MOI that minimizes double-transduction.
  • Cells can be treated with detergents as described herein and incubated with a detectable, e.g. fluorescent, ligand to label functional protein mutants encoded by favourable gene mutants from the library.
  • Cells can then be sorted, e.g. using FACS, to select the cells containing the favourable library-encoded mutants. Multiple rounds of selection may be performed in succession to enrich the desired population.
  • the library gene pool of the selected population can be isolated, e.g. via genomic extraction and PCR, and individual mutants isolated for characterization.
  • the library gene pool can then be used as the template for a subsequent generation of random mutagenesis of the target gene for further rounds of selection.
  • a third-generation lentiviral transfer plasmid, pLenti-displ (Figure 2), was designed for the generation, lentiviral transduction, and mammalian expression of a GPCR library.
  • the plasmid contained the following components: HIV-1 5' and 3' long terminal repeats (HIV-1 LTRs), HIV-1 psi (i ) packaging signal, central polypurine tract (cPPT), cytomegalovirus promoter (CMV), Bacillus subtilis levansucrase (SacB) (flanked by BamHI and Nhel restriction sites), streptactin II (strep) tag, internal ribosome entry site (IRES), mCherry, woodchuck hepatitis virus post- transcriptional regulatory element (wPRE), Ampicillin resistance gene (AmpR) and a ColEl origin of replication (oriC).
  • the human VIA DNA was ligated into lentiviral transfer plasmid pLenti-displ-sac
  • HEK293T cells were transfected with a third-generation lentivirus system comprised of 4 plasmids: Two packaging plasmids (pMDL, pRSV-rev), an envelope plasmid (pCMV- VSV-G), and the transfer plasmid (pLenti-displ).
  • pMDL Two packaging plasmids
  • pRSV-rev an envelope plasmid
  • pCMV- VSV-G envelope plasmid
  • pLenti-displ transfer plasmid
  • HEK293T cells were seeded at 400 000 cells/mL in 10 mL DMEM supplemented with 1% L-glutamine and penicillin-streptomycin (Thermo) and 10% foetal bovine serum (FBS) (Thermo) in a 10 cm2 dish and incubated for 24 h at 37°C with 5% CO2 and 85% humidity for next day transfection.
  • DMEM fetal bovine serum
  • the transfection mixture was prepared as follows: 5.2 pg pMDL, 2 pg pRSV-rev, 2.8 pg pCMV-VSV-G, 8 pg pLenti-displ, 3 mL Opti-MEMTM (Gibco), 50 pL lipofectamine 2000 (Invitrogen), 7 mL FreestyleTM 293 Expression Medium (Gibco).
  • the DMEM was aspirated and incubated with transfection mixture for 48 h.
  • a 125 mL flask of 293-F cells was prepared at 200 000 cells/mL in 20 mL for next day transduction.
  • Ligated vector was transformed via electroporation into electrocompetent DH5a cells (Invitrogen), which were recovered for lh at 37°C in SOC media, before 100 pg/mL ampicillin was added and the culture was incubated at 37°C overnight.
  • the efficiency of the ligation and transformation i.e. transformed cells per mL of culture was estimated using serial dilutions of transformed culture on a 7% sucrose LB agar plate. The following day, the DNA was harvested from this culture using the PureLinkTM HiPure Plasmid Maxiprep kit. The purified DNA was used for lentivirus production.
  • VIA libraries were transduced at various dilutions to obtain a desirable MOI.
  • HEK293T cells were seeded at 400 000 cells/mL in 25 mL in a 20 cm2 dish for next day transfection.
  • the transfection mixture was prepared as follows: 10.4 pg pMDL, 4 pg pRSV- rev, 5.6 pg pCMV-VSV-G, 16 pg pLenti-displ-VlA library, 6 mL Opti-MEMTM (Gibco), 100 pL lipofectamine 2000 (Invitrogen), 19 mL FreestyleTM 293 Expression Medium (Gibco).
  • the HEK293T cells were aspirated and incubated with transfection mixture for 48h.
  • 4 flasks of 293-F cells were prepared at 200 000 cells/mL in 20mL for next day transduction.
  • 25 mL viruscontaining media was harvested, and filtered through a 0.45 pm filter.
  • 25 pL of polybrene was added to the viral media.
  • the 293-F cultures were spun down, aspirated, and incubated for 8 hours with 4 dilutions of viral media (1, 0.5, 0.2, 0.1), each totalling 12 mL.
  • sorting sample 20 mL cells (sorting sample) were spun down and resuspended in 4 L media (with fluorescent ligand), while 5 mL cells (competition sample) were resuspended in 1 mL media with fluorescent ligand and excess competitor; VIA conditions used 50 nM A647-PhAcALVP ⁇ 10 pM SR49059 (Serradeil-Le Gal et al., 1993) . Samples were incubated at room temperature for lh with inversion, then filtered through filter mesh in preparation for flow cytometry.
  • Selections were performed using a BD FACSAria III (BD-Biosciences), gating for viable cells (DAPI-), mCherry-positive, and high, specific, ligand fluorescence. Selection stringency was gradually increased each round until the cells in the top ⁇ 1% of ligand. Selected cells were recovered in 5 mL FreestyleTM 293 Expression Medium (Gibco) for lh before being spun down and seeded at 500 000 cells/mL for culturing. Following the rounds of the selection, the genome of the final population was extracted using the Arcturus® PicoPure® DNA extraction kit (ThermoFisher). Two consecutive PCRs were performed on the extraction - the first using lentiviral primers and the second using receptor specific primers.
  • PCRs were performed using VELOCITY DNA Polymerase (Bioline). The extracted GPCR genes were re-ligated into the respective pLenti vectors to isolate and characterise individual GPCR clones. 46 VIA clones were isolated and screened.
  • HEK293T cells were seeded (170 000 cells/well) in a 24-well plate for next day transfection.
  • Transfections of VIA mutants #1-23 & wild-type in pcDNA3.1-Zeo(+)-FLAG used 0.5 pg of DNA and 2 pL of lipofectamine 2000 (Invitrogen) per well.
  • transfection media was aspirated from wells and cells were resuspended in phenol red-free DMEM (Gibco) and aliquoted into a 96 well U-bottom plate at 50 000 cells per well.
  • 50 nM A647-PhAcALVP and lOpM SR49059 was added to each mutant in duplicate.
  • HEK293T cells were seeded (1 million cells/well) in a 6-well plate for next day transfection.
  • Transfections used lpg of DNA and 4pL of lipofectamine 2000 (Invitrogen) per well. 48 hours after transfection, transfection media was aspirated from wells and cells were resuspended in phenol red-free DMEM (Gibco) and aliquoted into a 96 well U-bottom plate with relevant concentrations of A647-PhAcALVP. 10 pM SR49059 was used as competition.
  • HEK293T cells were seeded (1 million cells/well) in a 6-well plate for next day transfection. Transfections used lpg of DNA and 4pL of lipofectamine 2000 (Invitrogen) per well. 48 hours after transfection, transfection media was aspirated from wells and cells were resuspended in phenol red-free DMEM (Gibco) and aliquoted into a 96 well V-bottom plate with relevant concentrations of A647-PhAcALVP. 10 pM SR49059 was used as competition. Plate was gently shaken on an orbital shaker for lh at RT then analysed using a Cytoflex S flow cytometer (Beckman Coulter). Mean A647 fluorescence from competition sample (+10 pM SR49059) was subtracted from the A647- PhAcALVP-only samples to give specific binding data. All experiments were performed in triplicate and regression analyses were performed using GraphPad Prism.
  • HEK293T cells were seeded at 1 million cells/well in a 6-well plate. Cells were transfected the next day with a VIA mutant or wild-type using mammalian expression vector pcDNA3.1-Zeo(+), with 2 pg DNA and 8 pL lipofectamine added per well. 48 hours later, cells were harvested, spun down, flash frozen, and stored at -80°C until the day of the experiment.
  • lysis buffer 50 mM Tris, 500 mM NaCI, 1 mM PMSF, 5 mM TCEP, DNAse, complete protease inhibitor (Roche), 1% DDM, 0.03% CHS, pH 7.4.
  • 500 pL lysis buffer per pellet was used for thermostability assays or 250 pL per pellet for expression assays.
  • Cells were solubilised at 4°C for 90 minutes before being ultracentrifuged at 4°C for lh at 100 000 x g.
  • 100 pL of cell supernatant was heated on a PCR cycler for 20 minutes prior to HPLC analyses.
  • HEK293T cells we seeded at 20 000 cells per well in a 96-well plate (Corning).
  • Cells were transfected the following day with a VIA mutant or wild-type VIA via pcDNA3.1-Zeo(+)- HIBit, as well as pCRE-Pgal, using 0.25 pg DNA per plasmid and 0.5 pL Lipofectamine 2000 (Invitrogen) per well. The next day cells were incubated with increasing concentrations of AVP for VIA, using 5 pM forskolin as a positive control, and media as a negative control. Cells were incubated for 6 h at 37 °C.
  • cell supernatant was aspirated and 96-well plates were frozen at -80°C
  • 25 pL assay buffer 1 (10 mM Na2HPO4, 200 nM MgSO4, 10 pM MnCI2, pH 8.0) and placed on an orbital shaker for 10 minutes.
  • Cell supernatant was aspirated, then cells were treated with 100 pL assay buffer 2 (100 mM Na2HPO4, 2 mM MgSO4, 0.1 mM MnCI2, 0.5% Triton X-100, 40 mM B-mercaptoethanol, pH 8.0) and placed on an orbital shaker for 10 minutes.
  • ROCHE chlorophenolred B-D galactopyranoside
  • HEK293T cells were seeded at 1 million cells/well in a 12-well plate. Cells were transfected the next day with a VIA mutant or wild-type using mammalian expression vector pcDNA3.1-Zeo(+), with 1 pg DNA and 4 pL lipofectamine added per well. The following day, cells were plated out in a Poly-D-lysine-treated 96-well plate at 30 000 cells per well, 24-wells per construct.
  • a gene delivery system was required that could stably incorporate only a single GPCR mutantencoding gene into each cell of a large culture.
  • Various recombinant viruses are commonly used to deliver exogenous genes to mammalian cells, including adenoviral vectors, adeno-associated viral vectors and lentiviral vectors. Viral transduction can be titrated so that on average, transduced cells will be infected by single recombinant viral particles, thus allowing single mutants to be delivered efficiently.
  • Lentiviral vectors were chosen to deliver the GPCR gene libraries to mammalian cells because being an RNA virus, the transgene is stably integrated into the genomic DNA of transduced cells. Lentiviruses also boast a smaller plasmid size, a relatively large genomic capacity, and the ability to infect most cells at various cell cycle stages.
  • a custom third-generation lentiviral transfer plasmid, pLenti-displ, was constructed comprising a viral cassette containing HIV-1 5' and 3' long terminal repeats and a psi packaging signal, a Cytomegalovirus promoter, a cloning site flanking the negative selection marker sacB (Bacillus subtilis levansucrase), and an internal ribosome entry site (IRES) driving expression of the fluorescent protein mCherry (see Figure 2).
  • a lentivirus-driven method for selecting high-expressing proteins in mammalian cells is described herein, by selecting for variant proteins that express at high levels in human cells and are resistant to detergents (see Figure 1).
  • this method can be can be used to: (1) select for protein mutants that are resistant to detergents and; (2) allow the engineering of membrane proteins no matter the sub-cellular location, by enabling access of external drugs/ligands to the inside of the cell ( Figure 3).
  • the CSC of a detergent is the amount required to disrupt a membrane system into a predominantly micellar dispersion (Chattopadhyay et al., 2015; Prive, 2007).
  • the CSC is an intrinsic property of the detergent selected, as well as solubilization conditions, including cell membrane concentration, temperature and time. This is different to the critical micelle concentration (CMC) of a detergent, which is the minimal concentration of a detergent that will spontaneously form a micelle.
  • CMC critical micelle concentration
  • the proportion of particles that maintained the FSC/SCC properties of the untreated cells was identified and loss of cells in this gate as a function of detergent concentration was tracked. It was demonstrated that HEK293F cells were tolerant of most LMNG concentrations tested, and maintained cell-like scatter profiles after treatment with up to 0.025%w/v LMNG ( Figure 4). With DDM treatment however, cells were lysed in at DDM concentrations down to 0.003125% w/v. Often the transition from membrane to micelle occurs over a sharp concentration range (Prive, 2007), which is what we observed with DDM. Nonetheless, a window of sub-CSC DDM treatment concentrations was found where cell viability was maintained, as in the scenario depicted in Figure 4A.
  • variant 1 is wild type (WT) rat NTR
  • variant 2 is a constitutively active mutant of rat NTR (with 18 amino acid substitutions compared to variant 1).
  • Constitutive activity is defined as ligand independent activity caused by G protein coupling in the absence of an agonist. This means that the receptor is regularly sampling an active conformation and thus would be expected to be less “stable” and more sensitive to detergent (Krumm et al., 2015).
  • HEK293F cells were transfected with either NTR variant 1 or NTR variant 2 and incubated with a saturating concentration of fluorescently labeled neurotensin peptide (TAMRA-NTS-8-13), with, or without 10 pM of unlabeled neurotensin peptide (NTS8-13) as a competitor for 1.5 h. Cells were then washed, and incubated with increasing concentrations of detergent for 10 minutes at 20°C. Detergent was removed, and cells were analysed with flow cytometry.
  • TAMRA-NTS-8-13 fluorescently labeled neurotensin peptide
  • NTS8-13 unlabeled neurotensin peptide
  • TAMRA fluorescence Specific fluorescent ligand binding
  • HEK293F cells were transduced with dopamine receptor D2 (D2), or serotonin receptor 5-HT2A, using the aforementioned pLenti-displ lentivirus.
  • D2 dopamine receptor D2
  • L0002RED serotonin receptor 5-HT2A
  • Heatmap scatterplots of cells treated with and without digitonin Figure 6) describe how cells treated with digitonin exhibit a reduced forward scatter (FSC) profile, i.e. size, compared to whole cells, but remain distinguishable and selectable from non-cell particles.
  • FSC forward scatter
  • Figure 6C shows that cells transduced with D2 receptor do not display specific binding on the cell surface, evidence by no difference compared to negative control non-specific binding (NSB) cells, which were treated with 10 pM spiperone as competition. However, when these cells were treated with digitonin ( Figure 6D), a small population of cells (likely those expressing the transgene) exhibited specific binding of ligand when compared to NSB control cells. Notably, these cells would be able to be sorted using FACS.
  • genomic extraction can be performed on the selected population of cells, and the selected receptor genes isolated using PCR, as described previously. Although the nuclear membrane is contains the presence of cholesterol and is potentially affected by exposure to detergent of detergent-like compounds, genomic DNA remains encased in the nucleus.
  • EXAMPLE 4 DIRECTED EVOLUTION OF HIGH-EXPRESSING ENGINEERED VIA VARIANTS
  • the following disclosure provides exemplification of the use of a lentivirus-based gene delivery system to stably incorporate only a single protein variant-encoding gene into each cell, and of cell sorting technology such as FACS to select variant proteins of interest that can be generated using the above-described methods.
  • the coding region of wild-type VIA was ligated into pLenti-displ encoding the mCherry fluorescent protein, and the resulting plasmid used to generate lentiviral particles to transduce HEK 293-F cells with a VlA-encoding LV.
  • a week after transduction over 47 % of the cell population expressed mCherry, indicating that the LV particles were produced and able to transduce cells (Figure 7).
  • Alexa647-PhAcALVP a linear, peptide VIA antagonist was used to determine if VIA protein was expressed in the transduced cells.
  • mCherry positive cells exhibited significant binding of Alexa647-PhAcALVP, which was able to be competed with excess VIA antagonist SR49059 (Figure 7).
  • mCherry negative cells displayed no binding of Alexa647-PhAcALVP over baseline, demonstrating that pLenti-displ-VlA transduction resulted in robust transgene expression.
  • a randomly mutated library of the VIA gene was prepared and ligated into pLenti- displ, generating pLenti-displ-VlA-EPl.
  • pLenti-displ-VlA-EPl virus was produced, as described above, and applied to populations of 4 million 293-F cells at various dilution factors (undiluted, 0.5, 0.2, 0.1) (Figure 8) with the aim of achieving a multiplicity of infection (MOI) that minimizes doubletransductions.
  • MOI multiplicity of infection
  • a MOI of approximately 0.3 is desired, which equates to a P[0] of 0.74 and a P[>1] of 0.037
  • the HEK 293-F culture treated with a 0.2 dilution of pLenti-displ-VlA-EPl virus resulted in a transduction efficiency of 20.6 % (as judged by the number of viable positive cells) and was chosen for FACS selection.
  • the VIA EPl library-expressing culture was subjected to three consecutive rounds of FACS selection for high A647-PhAcALVP binding to enrich the population of cells for high cellsurface expression of VIA.
  • VIA EP1AS3 genomic DNA was used as a template for a second round of mutagenesis and library preparation as described above, generating the VIA EP2 cell population, which was subjected to four consecutive rounds of FACS selection for high A647-PhAcALVP fluorescence.
  • the mean A647-PhAcALVP cell binding of the final population (VIA EP2AS4) was over 10-fold higher than that of cells expressing wild-type VIA ( Figure 9A).
  • VIA clones were isolated via genomic DNA extraction, PCR and recloning (23 from VIA EP1AS3 and 23 from VIA EP2AS4) and transfected into 293-T cells for a singleconcentration A647-PhAcALVP binding screen ( Figure 10). 14 of the highest expressing clones were sequenced, transfected into cells for A647-PhAcALVP saturation binding assays ( Figures 9B and 11) and Fluo-8 AM calcium mobilization assays ( Figures 9C and 12). 11 VIA mutants displayed significantly increased cell surface expression compared to VIA wild-type ( Figure 9B).
  • AVP stimulation of cells expressing the selected VIA mutants revealed that 8 of the clones could activate Ca2+ mobilization in a similar way to VIA wild-type, whereas three (V1A#4, V1A#11, and V1A#34) were signaling incapable.
  • EXAMPLE 5 SELECTION OF A GPCR LIBRARY FOR HIGH-EXPRESSING MUTANTS RESISTANT TO SUB-CSC DETERGENT
  • the VIA EP2 AS3 library population was chosen as a test case for selecting detergent insensitive mutants.
  • 40 million cells from the VIA EP2 AS3 culture were treated with 0.003% DDM in freestyle media for 10 minutes at 20°C with gentle inversion. Cells were then centrifuged at 500 x g for two minutes, resuspended in 5 mL freestyle media, centrifuged again, and resuspended in ligand mixes as follows: 50 nM A647-PhAcALVP for the sorting sample, and 50 nM A647-PhAcALVP with additional 10 pM SR49059 to determine non-specific binding. Cells were incubated with ligand mixes for one hour at 20°C with gentle inversion. Prior to sorting, DAPI was added to cells to fluorescently label non-viable cells.
  • FIG. 13 shows the ligand binding (A647 fluorescence) of cells treated with and without detergent.
  • the detergent treated sample (0.003% DDM) exhibits a reduction in ligand binding when compared to the non-treated sample (0% DDM), which was expected as detergent treatment would reduce the binding capacity of the receptor.
  • the non-treated sample 0% DDM
  • the non-specific binding sample which was treated with additional 10 pM SR49059 as competitor, there is a population that was able to bind ligand with specificity despite being challenged with detergent treatment.
  • the top 0.5 % of A647 fluorescent cells in the populations treated with 0.003% DDM population were sorted, indicated in Figure 13 with the dashed square, in order to capture the highest ligand binders from the detergent-challenged sample. At least 20 000 cells were obtained from the sort, and sorted cells were centrifuged at 500 x g for 2 minutes, before being resuspended in DMEM supplemented with 1% L-glutamate and 10% Fetal Bovine Serum (FBS) for recovery.
  • FBS Fetal Bovine Serum
  • ViA DDM AS2 Following two detergent-treated sorts (VIA DDM AS2) as described above, selected ViA-encoding clones were isolated via PCR of genomically extracted DNA from the sorted cells. Single selected VIA clones were ligated into pCSC-HA-ALFA-SacB-IRES-mCherry as in EXAMPLE 4. Eight of these selected VIA clones were randomly selected to compare to VIA wild-type (wt VIA) . HEK 293F cells were transfected with receptor constructs and 24 h later subjected to a ligand binding screen to determine cell-surface expression.
  • Cells were treated with 50 nM A647- PhAcALVP, with samples containing additional 10 pM SR49059 used to determine non-specific binding. Cells were incubated with ligand for 1 hour at 20°C with gentle inversion, before adding DAPI to fluorescently label non-viable cells. Cells were then analysed using flow cytometry, gating for single, mammalian cell-sized, DAPI-negative, mCherry-positive particles, as well as detecting A647 fluorescence. Substantial increases in cell-surface expression were observed for five of the eight selected VIA clones, with the best performing clone #22 exhibiting an approximate 9-fold signal increase over VIA wild-type ( Figure 14).
  • EXAMPLE 6 SELECTION OF GPCRS IN PERMEABILISED CELLS FOR THE BINDING OF RECEPTOR CONFORMATIONALLY SELECTIVE ANTIBODY TO INTRACELLULAR EPITOPES
  • HEK293F cells were transduced with an adrenergic alpha 1A (alA) receptor that had a chimeric swap made with the intracellular ends of transmembrane 5 & 6 and intracellular loop 3 of the kappa opioid receptor (KOR); termed from this point forward as the alA-KOR mutant.
  • This alA-KOR is consequently capable of binding to nanobody 6 (Nb6) that recognizes the inactive state conformation (stabilised by receptor inverse agonist binding and de-stabilised by receptor agonist binding) of the extracellular ends of transmembrane 5 & 6 of the KOR (Che et al., 2020).
  • Figure 16B shows that cells transduced with the alA-KOR mutant do not display specific binding on the cell surface, evidence by no difference compared to negative control nonspecific binding (NSB) cells (non-transduced cells).
  • Nb negative control nonspecific binding
  • Figure 16B when these cells were treated with GDN ( Figure 16B), there was specific binding of Cy5-Nb6 when compared to NSB control cells.
  • the amount of fluorescent staining observed was proportional to the conformational state of the receptor population, with higher fluorescent events in the conditions with the inverse agonist than without ligand, and a complete abolition of Nb6 staining seen in the presence of agonist (Figure 16C).
  • Nb6-Fc fusion conformational dependent Nb6 staining could be detected when purified Nb6 was used in either a format where it was directly fluorescently labeled (Cy5-Nb6) or it was in the format of an Fc-fusion & co-incubated with a fluorescent secondary antibody (Nb6-Fc fusion).
  • Table 1 Detergents and detergent like compounds useful in the methods disclosed herein.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Hematology (AREA)
  • Zoology (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Toxicology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Food Science & Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Endocrinology (AREA)
  • Neurology (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Virology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des procédés pour la sélection ou l'évolution dirigée de protéines pour la technique d'ingénierie de protéines ayant des caractères souhaités. L'invention concerne des procédés de sélection de variants (tels que des variants stables au détergent) d'une protéine d'intérêt ayant une activité souhaitée à partir d'une bibliothèque de séquences d'acides nucléiques exprimées codant pour des variants de la protéine d'intérêt.
PCT/AU2023/050493 2022-06-06 2023-06-06 Procédés de production de variants polypeptidiques WO2023235921A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2022901541 2022-06-06
AU2022901541A AU2022901541A0 (en) 2022-06-06 Methods for producing polypeptide variants

Publications (1)

Publication Number Publication Date
WO2023235921A1 true WO2023235921A1 (fr) 2023-12-14

Family

ID=89117206

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2023/050493 WO2023235921A1 (fr) 2022-06-06 2023-06-06 Procédés de production de variants polypeptidiques

Country Status (1)

Country Link
WO (1) WO2023235921A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014140586A2 (fr) * 2013-03-15 2014-09-18 Medical Research Council Protéines mutantes et leurs procédés de production

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014140586A2 (fr) * 2013-03-15 2014-09-18 Medical Research Council Protéines mutantes et leurs procédés de production

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ALMO STEVEN C, LOVE JAMES D: "Better and faster: improvements and optimization for mammalian recombinant protein production", CURRENT OPINION IN STRUCTURAL BIOLOGY, ELSEVIER LTD., GB, vol. 26, 1 June 2014 (2014-06-01), GB , pages 39 - 43, XP093116753, ISSN: 0959-440X, DOI: 10.1016/j.sbi.2014.03.006 *
CHE T ET AL.: "Nanobody-enabled monitoring of kappa opioid receptor states", NAT COMMUN, vol. 11, 2020, pages 1145, XP055965064, DOI: 10.1038/s41467-020-14889-7 *
MANGLIK AASHISH ET AL: "Nanobodies to Study G Protein-Coupled Receptor Structure and Function", ANNUAL REVIEW OF PHARMACOLOGY AND TOXICOLOGY, VOL 57 ANNUAL REVIEWS, 4139 EL CAMINO WAY, PO BOX 10139, PALO ALTO, CA 94303-0897 USA SERIES : ANNUAL REVIEW OF PHARMACOLOGY AND TOXICOLOGY (0362-1642(PRINT)), 7 December 2016 (2016-12-07), pages 19 - 37, XP002782170 *

Similar Documents

Publication Publication Date Title
US11390653B2 (en) Amino acid-specific binder and selectively identifying an amino acid
JP4262778B2 (ja) 生体分子相互作用を検出するためのタンパク質断片相補性アッセイ
JP2022502045A (ja) ロード可能な検出分子を使用したハイスループットのエピトープ同定及びｔ細胞受容体特異性決定
Zhang et al. Sensing of biomolecular interactions using fluorescence complementing systems in living cells
US20160146786A1 (en) Method of monitoring cellular trafficking of peptides
US20150010932A1 (en) Methods for assaying protein-protein interactions
US20220404357A1 (en) GENETICALLY ENCODED FLUORESCENT SENSORS FOR DETECTING LIGAND BIAS AND INTRACELLULAR SIGNALING THROUGH cAMP PATHWAYS
KR20210112347A (ko) 단백질 분해의 조정 가능한 제어를 위한 단백질 스위치의 데노보 설계
AU2021269475B2 (en) High-throughput screening methods to identify small molecule targets
Braun et al. High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology
Chojnacki et al. Investigation of HIV‐1 assembly and release using modern fluorescence imaging techniques
Makhija et al. Versatile labeling and detection of endogenous proteins using tag-assisted split enzyme complementation
US6747135B1 (en) Fluorescent dye binding peptides
US20160068861A1 (en) Method for detecting protein stability and uses thereof
Song et al. Monitoring G protein-coupled receptor activation using an adenovirus-based β-arrestin bimolecular fluorescence complementation assay
CN104844691A (zh) Sox2蛋白肽适配子及其鉴定
WO2023235921A1 (fr) Procédés de production de variants polypeptidiques
Lau et al. Rapid HIV-1 capsid interaction screening using fluorescence fluctuation spectroscopy
Raeeszadeh‐Sarmazdeh et al. Identifying stable fragments of Arabidopsis thaliana cellulose synthase subunit 3 by yeast display
US9400249B2 (en) Detection of biopolymer interactions, cancer cells, and pathogens using split-supercharged GFP
Sjöström et al. Tuning the binding interface between Machupo virus glycoprotein and human transferrin receptor
Leonhardt et al. CryoEM structures of the human HIV-1 restriction factor SERINC3 and function as a lipid transporter
KR102074590B1 (ko) 자가포식체 탐침용 프로브 및 이를 이용한 탐침 방법
WO2004027057A1 (fr) Procede d'analyse d'une proteine localisee dans un organite et matieres servant a cette analyse
Braun et al. High-throughput characterization of 309 photocrosslinker-bearing ASIC1a variants maps residues critical for channel function and pharmacology

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23818636

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)