Systems and methods for protein expression

Abstract

To provide a system and a method for protein expression.SOLUTION: The present disclosure provides a system for the expression of target protein in conjunction with enhancer protein. The enhancer protein may be a viral protein that blocks nucleocytoplasmic transport. Also provided are polynucleotides, vectors, and cells comprising target protein and enhancer protein nucleic acid sequences. In one aspect, the disclosure provides a system for recombinant expression of a target protein in eukaryotic cells that includes one or more vectors. The vectors (or a vector) have a first polynucleotide encoding the target protein and a second polynucleotide encoding an enhancer protein.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 901,043, filed on September 16, 2019, and U.S. Provisional Patent Application No. 62 / 970,628, filed on February 5, 2020, the entire contents of each of these provisional applications being incorporated herein by reference for all purposes.

[0002] Incorporation of Sequence Listing The content of the text file attached hereto and electronically submitted is incorporated herein by reference in its entirety: computer - readable format copy of the sequence listing (file name: EXCI_001_02WO_SeqList_ST25.txt, recording date: September 15, 2020, file size: 84.8 kb).

Background Art

[0003] Background Recombinant expression of proteins in eukaryotic cells grown in culture is useful in scientific research and medicine. Recombinant - produced proteins (such as antibodies, enzymes, G - protein - coupled receptors (GPCRs), secreted proteins, ion channels, viral proteins, and growth factors, etc.) are used within the pharmaceutical industry as therapeutic agents (e.g., antibodies and other biological drugs) and as critical resources for analytical methods in order to develop new drugs (e.g., small - molecule discovery). In addition to these uses within the pharmaceutical industry, recombinant - produced mammalian proteins are increasingly used in the food industry (e.g., for so - called clean meat production). For many recombinant proteins, achieving the expression of the recombinant protein in a functional form remains difficult.

[0004] The need for compositions and methods useful in the production of recombinant proteins remains unmet.

Summary of the Invention

Means for Solving the Problem

[0005] Summary The inventors recognized that co-expression of a target protein and a certain enhancer protein improves the protein produced by recombination. In various embodiments, the disclosed compositions and methods exhibit one or more of the following advantages over the prior art: (1) the disclosed compositions and methods increase the protein expression (yield) of the target protein in a cell line (e.g., a eukaryotic cell line); (2) the disclosed compositions and methods control the regulation of the expression of the target protein; (3) the disclosed compositions and methods express a target protein that exhibits improved properties (e.g., reduced misfolding, altered activity, inaccurate post-translational modification and / or toxicity); (4) the disclosed compositions and methods increase the accurate folding and / or high yield of the recombinant protein; (5) the disclosed compositions and methods improve the performance of downstream activation pathways (e.g., GPCR signaling); and / or (6) co-expression of the enhancer protein does not affect the functionality of the target protein and / or the downstream metabolism of the cell. Some embodiments do not exhibit any of these advantages, exhibit some of them, or exhibit all of them. Therefore, the present invention is not limited by these listed advantages.

[0006] In one aspect, the present disclosure provides a system for recombinant expression of a target protein in a eukaryotic cell comprising one or more vectors. The vector(s) has a first polynucleotide encoding the target protein and a second polynucleotide encoding an enhancer protein. The enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or the enhancer protein is selected from the group consisting of picornavirus leader (L) protein, picornavirus 2A protease, rhinovirus 3C protease, herpes simplex virus (HSV) ICP27 protein, and rhabdovirus matrix (M) protein. The first polynucleotide and the second polynucleotide are operably linked to one or more promoters.

[0007] In another aspect, the present disclosure provides a eukaryotic cell for the expression of a target protein, the cell comprising an exogenous polynucleotide encoding an enhancer protein. The enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or the enhancer protein is selected from the group consisting of picornavirus leader (L) protein, picornavirus 2A protease, rhinovirus 3C protease, coronavirus ORF6 protein, Ebola virus VP24 protein, Venezuelan equine encephalitis virus (VEEV) capsid protein, herpes simplex virus (HSV) ICP27 protein, and rhabdovirus matrix (M) protein. The exogenous polynucleotide is operably linked to a promoter (optionally, a native promoter or an exogenous promoter). In yet another aspect, the present disclosure provides a method for the recombinant expression of a target protein, the method comprising introducing into the eukaryotic cell a polynucleotide encoding the target protein, operably linked to a promoter. In yet another aspect, the present disclosure provides a method for the recombinant expression of a target protein, the method comprising introducing into the eukaryotic cell the vector system of the present disclosure. In yet another aspect, the present disclosure provides a cell produced by introducing the vector system (or vector) of the present disclosure into a eukaryotic cell. In yet another aspect, the present disclosure provides a protein expressed by introducing the vector system (or vector) of the present disclosure into a eukaryotic cell. In yet another aspect, the present disclosure provides a method for expressing a target protein in a eukaryotic cell, the method comprising introducing into the eukaryotic cell a polynucleotide encoding the target protein (a polynucleotide operably linked to a promoter). The method utilizes co-expression of an enhancer protein to enhance the expression level, solubility, and / or activity of the target protein.The enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT) and / or the enhancer protein is selected from the group consisting of picornavirus leader (L) protein, picornavirus 2A protease, rhinovirus 3C protease, coronavirus ORF6 protein, Ebola virus VP24 protein, Venezuelan equine encephalitis virus (VEEV) capsid protein, herpes simplex virus (HSV) ICP27 protein, and rhabdovirus matrix (M) protein.

[0008] In another aspect, the present disclosure provides a method for generating an antibody against a target protein, the method comprising immunizing a subject with a cell or a target protein produced using the system or method of the present disclosure. In yet another aspect, the present disclosure provides a method for antibody discovery by cell sorting, the method comprising preparing a solution comprising a labeled cell or a labeled target protein produced using the system or method of the present disclosure, and a population of recombinant cells, wherein the recombinant cells express a library of polypeptides each comprising an antibody or an antigen-binding fragment thereof, and selecting one or more recombinant cells from the solution by detecting the recombinant cells bound to the labeled cell or the labeled target protein. In a further aspect, the present disclosure provides a method for panning a phage display library, the method comprising mixing the phage display library with a cell or a target protein produced using the system or method of the present disclosure, and purifying and / or enriching members of the phage display library that bind to the cell or the target protein.

[0009] Further aspects and embodiments are provided by the following detailed disclosure. The invention is not limited by this summary. In embodiments of the present invention, for example, the following items are provided. (Item 1) A system for recombinant expression of a target protein in a eukaryotic cell, comprising one or more vectors, wherein the one or more vectors comprise a) a first polynucleotide encoding the target protein, and b) a second polynucleotide encoding an enhancer protein and i) the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or ii) the enhancer protein is selected from the group consisting of picornavirus leader (L) protein, picornavirus 2A protease, rhinovirus 3C protease, herpes simplex virus (HSV) ICP27 protein, and rhabdovirus matrix (M) protein, wherein the first polynucleotide and the second polynucleotide are operably linked to one or more promoters. (Item 2) The system according to item 1, wherein the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT). (Item 3) The system according to item 2, wherein the NCT inhibitor is a viral protein. (Item 4) The system according to any one of items 1 to 3, wherein the NCT inhibitor is selected from the group consisting of picornavirus leader (L) protein, picornavirus 2A protease, rhinovirus 3C protease, coronavirus ORF6 protein, Ebola virus VP24 protein, Venezuelan equine encephalitis virus (VEEV) capsid protein, herpes simplex virus (HSV) ICP27 protein, and rhabdovirus matrix (M) protein. (Item 5) The system according to item 4, wherein the NCT inhibitor is picornavirus leader (L) protein or a functional variant thereof. (Item 6) The system according to item 4, wherein the NCT inhibitor is a picornavirus 2A protease or a functional variant thereof. (Item 7) The system according to item 4, wherein the NCT inhibitor is a rhinovirus 3C protease or a functional variant thereof. (Item 8) The system according to item 4, wherein the NCT inhibitor is a coronavirus ORF6 protein or a functional variant thereof. (Item 9) The system according to item 4, wherein the NCT inhibitor is an Ebola virus VP24 protein or a functional variant thereof. (Item 10) The system according to item 4, wherein the NCT inhibitor is a Venezuelan equine encephalitis virus (VEEV) capsid protein or a functional variant thereof. (Item 11) The system according to item 4, wherein the NCT inhibitor is a herpes simplex virus (HSV) ICP27 protein or a functional variant thereof. (Item 12) The system according to item 4, wherein the NCT inhibitor is a rhabdovirus matrix (M) protein or a functional variant thereof. (Item 13) The system according to item 5, wherein the L protein is the L protein of Theiler's virus or a functional variant thereof. (Item 14) The system according to item 5, wherein the L protein shares at least 90% identity with SEQ ID NO: 1. (Item 15) The system according to item 5, wherein the L protein is the L protein of encephalomyocarditis virus (EMCV) or a functional variant thereof. (Item 16) The system according to item 5, wherein the L protein shares at least 90% identity with SEQ ID NO: 2. (Item 17) The system according to item 5, wherein the L protein is selected from the group consisting of the L protein of poliovirus, the L protein of HRV16, the L protein of mengovirus, and the L protein of sapovirus 2, or a functional variant thereof. (Item 18) The system according to any one of items 1 to 17, wherein the system comprises a single vector containing an expression cassette, and the expression cassette contains the first polynucleotide and the second polynucleotide. (Item 19) The system according to item 18, wherein the expression cassette contains a first promoter operably linked to the first polynucleotide and a second promoter operably linked to the second polynucleotide. (Item 20) The system according to item 18, wherein the expression cassette contains a shared promoter operably linked to both the first polynucleotide and the second polynucleotide. (Item 21) The system according to item 20, wherein the expression cassette contains a coding polynucleotide containing the first polynucleotide and the second polynucleotide linked by a polynucleotide encoding a ribosome skipping site, and the coding polynucleotide is operably linked to the shared promoter. (Item 22) The system according to item 20, wherein the expression cassette contains a coding polynucleotide, the coding polynucleotide encodes the enhancer protein and the target protein linked by a ribosome skipping site, and the coding polynucleotide is operably linked to the shared promoter. (Item 23) The expression cassette is configured for the transcription of a single messenger RNA encoding both the target protein and the enhancer protein, linked by a ribosome skipping site, and the translation of the messenger RNA results in the expression of the target protein and the L protein as separate polypeptides. The system according to any one of items 18 to 22. (Item 24) The system according to any one of items 1 to 23, comprising one vector. (Item 25) a) A first vector comprising the first polynucleotide operably linked to a first promoter; b) A second vector comprising the second polynucleotide operably linked to a second promoter. The system according to any one of items 1 to 17, comprising the above. (Item 26) The system according to any one of items 1 to 17 or item 25, comprising two vectors. (Item 27) The system according to any one of items 1 to 26, wherein either or both of the first polynucleotide and the second polynucleotide are operably linked to an internal ribosome entry site (IRES). (Item 28) The system according to any one of items 1 to 27, wherein at least one of the one or more vectors comprises a T7 promoter configured for the transcription of either or both of the first polynucleotide and the second polynucleotide by T7 RNA polymerase. (Item 29) The system according to any one of items 1 to 28, wherein at least one of the one or more vectors comprises a polynucleotide sequence encoding T7 RNA polymerase. (Item 30) a) A first polynucleotide encoding a target protein; b) A second polynucleotide encoding an enhancer protein. A vector for recombinant expression of a target protein in eukaryotic cells, comprising i) the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or ii) the enhancer protein is selected from the group consisting of picornavirus leader (L) protein, picornavirus 2A protease, rhinovirus 3C protease, coronavirus ORF6 protein, Ebola virus VP24 protein, Venezuelan equine encephalitis virus (VEEV) capsid protein, herpes simplex virus (HSV) ICP27 protein, and rhabdovirus matrix (M) protein, A vector, wherein the first polynucleotide and the second polynucleotide are operably linked to at least one promoter. (Item 31) The vector according to item 30, wherein the expression cassette comprises a first promoter operably linked to the first polynucleotide and a second promoter operably linked to the second polynucleotide. (Item 32) The vector according to item 30, wherein the expression cassette comprises a shared promoter operably linked to both the first polynucleotide and the second polynucleotide. (Item 33) A eukaryotic cell for expression of a target protein, comprising an exogenous polynucleotide encoding an enhancer protein, a) the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or b) the enhancer protein is selected from the group consisting of picornavirus leader (L) protein, picornavirus 2A protease, rhinovirus 3C protease, coronavirus ORF6 protein, Ebola virus VP24 protein, Venezuelan equine encephalitis virus (VEEV) capsid protein, herpes simplex virus (HSV) ICP27 protein, and rhabdovirus matrix (M) protein, A eukaryotic cell in which the exogenous polynucleotide is operably linked to a promoter. (Item 34) The eukaryotic cell according to Item 33, wherein the polynucleotide is operably linked to an internal ribosome entry site (IRES). (Item 35) The eukaryotic cell according to Item 33 or Item 34, wherein the promoter is an inducible promoter. (Item 36) A method for recombinant expression of a target protein, comprising introducing a polynucleotide encoding the target protein, which is operably linked to a promoter, into the cell according to any one of Items 33 to 35. (Item 37) A method for recombinant expression of a target protein, comprising introducing the system according to any one of Items 1 to 29 or the vector according to any one of Items 30 to 32 into a eukaryotic cell. (Item 38) The method according to Item 36 or Item 37, wherein the target protein is a membrane protein. (Item 39) The method according to Item 38, wherein the localization of the membrane protein to the cell membrane is increased as compared to the localization observed when the membrane protein is expressed without the enhancer protein. (Item 40) A eukaryotic cell produced by introduction of the system according to any one of Items 1 to 29 or the vector according to any one of Items 30 to 32. (Item 41) A target protein expressed by introduction of the system according to any one of Items 1 to 29 or the vector according to any one of Items 30 to 32 into a eukaryotic cell. (Item 42) A method for expressing a target protein in a eukaryotic cell, comprising introducing a polynucleotide encoding the target protein, which is operably linked to a promoter, into the eukaryotic cell and including the step of The method enhances the expression level, solubility and / or activity of the target protein by utilizing co-expression of an enhancer protein, a) the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or b) the enhancer protein is selected from the group consisting of picornavirus leader (L) protein, picornavirus 2A protease, rhinovirus 3C protease, coronavirus ORF6 protein, Ebola virus VP24 protein, Venezuelan equine encephalitis virus (VEEV) capsid protein, herpes simplex virus (HSV) ICP27 protein and rhabdovirus matrix (M) protein. (Item 43) The co-expression of the enhancer protein comprises introducing into the eukaryotic cell a polynucleotide encoding the enhancer protein operably linked to a promoter, the method according to item 42. (Item 44) The step(s) of introducing comprises transfection of the eukaryotic cell with one or more DNA molecules, transduction of the eukaryotic cell with a single viral vector, and / or transduction of the eukaryotic cell with two viral vectors, the method according to item 42 or item 43. (Item 45) The target protein is a soluble protein, the system according to any one of items 1 to 29, the vector according to any one of items 30 to 32, the eukaryotic cell according to any one of items 33 to 35, the method according to any one of items 36 to 39 and 42 to 44, the eukaryotic cell according to item 40, and the target protein according to item 41. (Item 46) The target protein is a secreted protein, the system according to any one of items 1 to 29, the vector according to any one of items 30 to 32, the cell according to any one of items 33 to 35, or the method according to any one of items 36 to 44. (Item 47) The system according to any one of items 1 to 29, the vector according to any one of items 30 to 32, the eukaryotic cell according to any one of items 33 to 35, the method according to any one of items 36 to 39 and 42 to 44, the eukaryotic cell according to item 40, and the target protein according to item 41, wherein the target protein is a membrane protein. (Item 48) The system according to any one of items 1 to 29, the vector according to any one of items 30 to 32, the eukaryotic cell according to any one of items 33 to 35, the method according to any one of items 36 to 39 and 42 to 44, the eukaryotic cell according to item 40, and the target protein according to item 41, wherein the target protein is dopamine receptor 1 (DRD1), and optionally, the DRD1 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 19. (Item 49) The system according to any one of items 1 to 29, the vector according to any one of items 30 to 32, the eukaryotic cell according to any one of items 33 to 35, the method according to any one of items 36 to 39 and 42 to 44, the eukaryotic cell according to item 40, and the target protein according to item 41, wherein the target protein is cystic fibrosis transmembrane conductance regulator (CFTR), and optionally, the CFTR comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 18. (Item 50) The system according to any one of items 1 to 29, the vector according to any one of items 30 to 32, the eukaryotic cell according to any one of items 33 to 35, the method according to any one of items 36 to 39 and 42 to 44, the eukaryotic cell according to item 40, and the target protein according to item 41, wherein the target protein is C1-esterase inhibitor (C1-Inh), and optionally, the C1-Inh has at least 90% identity with the amino acid sequence of SEQ ID NO: 16 and comprises an amino acid sequence. (Item 51) The target protein is ITK, and optionally, the ITK comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 15. The system according to any one of items 1 to 29, the vector according to any one of items 30 to 32, the eukaryotic cell according to any one of items 33 to 35, the method according to any one of items 36 to 39 and 42 to 44, the eukaryotic cell according to item 40, and the target protein according to item 41. (Item 52) The target protein is NADase, and optionally, the NADase comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 20. The system according to any one of items 1 to 29, the vector according to any one of items 30 to 32, the eukaryotic cell according to any one of items 33 to 35, the method according to any one of items 36 to 39 and 42 to 44, the eukaryotic cell according to item 40, and the target protein according to item 41. (Item 53) A method for generating an antibody against a target protein, the method comprising immunizing a subject with the cell according to any one of items 33 to 35, the cell according to item 40, or the target protein according to item 41. (Item 54) The method according to item 53, further comprising isolating one or more immune cells that express an immunoglobulin protein specific for the target protein. (Item 55) The method according to item 53 or item 54, comprising generating one or more hybridomas from the one or more immune cells. (Item 56) The method according to any one of items 53 to 55, comprising cloning one or more immunoglobulin genes from the one or more immune cells. (Item 57) A method for antibody discovery by cell sorting, a) The cell according to any one of items 33 to 35, the eukaryotic cell according to item 40, or the target protein according to item 41, wherein the cell or the target protein is labeled, and b) A population of recombinant cells, wherein the recombinant cells express a library of polypeptides each containing an antibody or an antigen-binding fragment thereof, and Preparing a solution containing the same; Isolating one or more recombinant cells from the solution by selecting the labeled cells or the recombinant cells bound to the labeled target protein. A method comprising the steps of. (Item 58) A method for panning a phage display library, comprising: a) Mixing a phage display library with a eukaryotic cell according to any one of items 33 to 35, a eukaryotic cell according to item 40, or a target protein according to item 41; and b) Purifying and / or enriching members of the phage display library that bind to the cell or target protein. A method comprising the steps of. (Item 59) The eukaryotic cell according to any one of items 33 to 35 and 40, which is a human cell, an animal cell, an insect cell, a plant cell or a fungal cell. (Item 60) The eukaryotic cell according to any one of items 33 to 35, 40 and 59, which is a eukaryotic cell line. (Item 61) Bc HROC277, COS, CHO, CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV, VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO, 5P2 / 0-Ag14, HeLa, HEK293, HEK293-F, HEK293-H, HEK293-T, perC6 cells, Sf9 cells, Saccharomyces cells, Pichia cells or Schizosaccharomyces cells, which are eukaryotic cells according to any one of items 33 to 35, 40, 59 and 60. (Item 62) The eukaryotic cell according to item 60, wherein the eukaryotic cell line is a stable cell line. (Item 63) The system according to any one of items 1 to 29 and 45 to 52, wherein the one or more vectors are selected from the group consisting of adeno-associated virus (AAV) vectors, lentivirus vectors, retrovirus vectors, replication-competent adenovirus vectors, replication-deficient adenovirus vectors, herpesvirus vectors, baculovirus vectors, or non-viral plasmids. (Item 64) The system according to item 63, wherein at least one of the one or more vectors is an AAV vector. (Item 65) The vector according to any one of items 30 to 32, which is an adeno-associated virus (AAV) vector, a lentivirus vector, a retrovirus vector, a replication-competent adenovirus vector, a replication-deficient adenovirus vector, a herpesvirus vector, a baculovirus vector, or a non-viral plasmid. (Item 66) The vector according to item 65, which is an AAV vector. (Item 67) The system according to item 4, wherein the rhabdovirus matrix (M) protein is the M protein of vesicular stomatitis virus (VSV). (Item 68) The system according to item 67, wherein the M protein shares at least 90% identity with SEQ ID NO: 9. (Item 69) A system for recombinant expression of a target protein in a eukaryotic cell, comprising one or more vectors, wherein the one or more vectors are a) a first polynucleotide encoding the target protein, and b) A second polynucleotide encoding the L protein of encephalomyocarditis virus (EMCV), wherein, optionally, the L protein shares at least 90% identity with SEQ ID NO: 2, and the second polynucleotide and the system, wherein the first polynucleotide and the second polynucleotide are operably linked to one or more promoters. (Item 70) A system for recombinant expression of a target protein in a eukaryotic cell, comprising one or more vectors, wherein the one or more vectors a) a first polynucleotide encoding the target protein; and b) a second polynucleotide encoding the L protein of Theiler's virus, wherein, optionally, the L protein shares at least 90% identity with SEQ ID NO: 1, and the second polynucleotide and the system, wherein the first polynucleotide and the second polynucleotide are operably linked to one or more promoters. (Item 71) A system for recombinant expression of a target protein in a eukaryotic cell, comprising one or more vectors, wherein the one or more vectors a) a first polynucleotide encoding the target protein; and b) a second polynucleotide encoding picornavirus 2A protease, wherein, optionally, the picornavirus 2A protease shares at least 90% identity with SEQ ID NO: 7, and the second polynucleotide and the system, wherein the first polynucleotide and the second polynucleotide are operably linked to one or more promoters. (Item 72) A system for recombinant expression of a target protein in a eukaryotic cell, comprising one or more vectors, wherein the one or more vectors a) a first polynucleotide encoding the target protein; and b) A second polynucleotide encoding the M protein of vesicular stomatitis virus (VSV), wherein, if necessary, the M protein shares at least 90% identity with SEQ ID NO: 9, and the second polynucleotide and the first polynucleotide and the second polynucleotide are operably linked to one or more promoters. (Item 73) The system according to any one of Items 69 to 72, wherein the target protein is dopamine receptor 1 (DRD1), and wherein, if necessary, the DRD1 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 19. (Item 74) The system according to any one of Items 69 to 72, wherein the target protein is cystic fibrosis transmembrane conductance regulator (CFTR), and wherein, if necessary, the CFTR comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 18. (Item 75) The system according to any one of Items 69 to 72, wherein the target protein is C1-esterase inhibitor (C1-Inh), and wherein, if necessary, the C1-Inh comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 16. (Item 76) The system according to any one of Items 69 to 72, wherein the target protein is ITK, and wherein, if necessary, the ITK comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 15. (Item 77) The system according to any one of Items 69 to 72, wherein the target protein is NADase, and wherein, if necessary, the NADase comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 20.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0031] Detailed Description In the present disclosure, there are provided vector systems, vectors or eukaryotic cells useful in the co-expression of enhancer proteins and target proteins. In some embodiments, there is provided a system for recombinant expression of a target protein in a eukaryotic cell comprising one or more vectors. In some embodiments, the vector(s) has a first polynucleotide encoding a target protein and a second polynucleotide encoding an enhancer protein. The enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT) and / or the enhancer protein is selected from the group consisting of picornavirus leader (L) protein, picornavirus 2A protease, rhinovirus 3C protease, herpes simplex virus (HSV) ICP27 protein and rhabdovirus matrix (M) protein. The first polynucleotide and the second polynucleotide are operably linked to one or more promoters.

[0032] Without being bound by theory, the compositions and methods of the present disclosure prevent the cell regulatory mechanism from being activated in response to the expression of the recombinant target protein, thereby improving the yield and / or functionality of the target protein. The methods and systems of the present disclosure can inhibit or interfere with one or more cellular mechanisms including, but not limited to, (1) inhibition of transcription initiation, (2) inhibition of transcription termination and polyadenylation, (3) inhibition of mRNA processing and splicing, (4) inhibition of mRNA export, (5) inhibition of translation initiation, and (6) stress response (Figure 1).

[0033] Various embodiments are depicted in FIGS. 2A-2Y and Table 1. In some embodiments, the first vector comprises a polynucleotide encoding a target protein and the second vector comprises a polynucleotide encoding an enhancer protein. In other embodiments, a single vector comprises one or more polynucleotides encoding a target protein and an enhancer protein. The vector can comprise a single polynucleotide encoding both the target protein and the enhancer protein. Alternatively, two or more enhancer proteins and / or two or more target proteins are encoded by the vector(s).

[0034] Polynucleotide The present disclosure relates to recombinant polynucleotides for the expression of one or more target proteins and one or more enhancer proteins. The polynucleotide (or nucleic acid or nucleic acid molecule) can contain one or more genes of interest and is delivered to cells (e.g., eukaryotic cells) using the compositions and methods of the present disclosure. The polynucleotides of the present disclosure can include DNA, RNA, and DNA-RNA hybrid molecules. In some embodiments, the polynucleotide is isolated from a natural source; prepared in vitro using techniques such as PCR amplification or chemical synthesis; prepared in vivo, for example, by recombinant DNA technology; or prepared or obtained by any suitable method. In some embodiments, the polynucleotide can be of any shape (linear, circular, etc.) or topology (single-stranded, double-stranded, linear, circular, supercoiled, twisted, nicked, etc.). The polynucleotide can also include nucleic acid derivatives such as peptide nucleic acids (PNAs) and polypeptide-nucleic acid conjugates; nucleic acids having at least one chemically modified sugar residue, backbone, internucleotide linkage, base, nucleotide, nucleoside, or nucleotide analog or derivative; as well as nucleic acids having chemically modified 5' or 3' termini; and nucleic acids having two or more of such modifications. Not all linkages in the polynucleotide need to be identical.

[0035] Examples of polynucleotides include, but are not limited to, oligonucleotides (including oligonucleotides useful in antisense oligonucleotides, ribozymes, and RNA interference (RNAi)), aptamers, nucleic acids, artificial chromosomes, cloning vectors and constructs, expression vectors and constructs, gene therapy vectors and constructs, rRNA, tRNA, mRNA, mtRNA, and tmRNA, among others. In some embodiments, the polynucleotide is in vitro transcribed (IVT) mRNA. In some embodiments, the polynucleotide is a plasmid.

[0036] A polynucleotide is said to "encode" a protein if it contains a nucleic acid sequence that can be transcribed and translated (e.g., DNA → RNA → protein) or translated (RNA → protein) to produce an amino acid sequence corresponding to the amino acid sequence of the protein. In vivo (e.g., within a eukaryotic cell) transcription and / or translation is carried out by endogenous or exogenous enzymes. In some embodiments, transcription of the polynucleotides of the present disclosure is carried out by the endogenous polymerase II (polII) of eukaryotic cells. In some embodiments, the exogenous RNA polymerase is provided in the same or a different vector. In some embodiments, the RNA polymerase is selected from T3 RNA polymerase, T5 RNA polymerase, T7 RNA polymerase, and H8 RNA polymerase.

[0037] Exemplary polynucleotides according to the present disclosure include a "first polynucleotide" encoding a target protein; a "second polynucleotide" encoding an enhancer protein; and a "coding polynucleotide" encoding one or more target proteins, one or more enhancer proteins, and / or one or more isolated elements.

[0038] Target protein The polynucleotides according to the present disclosure can include nucleic acid sequences encoding one or more target proteins. The nucleic acid sequence encoding the target protein is referred to as the gene of interest (「GOI」). The target protein is any protein whose expression is desired. In some embodiments, the protein is a membrane protein. In some embodiments, when expressed in a reference expression system, the expression of the protein can cause cytotoxicity. In some embodiments, the protein is a protein with low-yield expression in a traditional expression system. In some embodiments, the expression or quality of the protein is significantly improved by expression according to the disclosed methods, for example, in combination with one or more enhancer proteins. In some embodiments, the target protein is an AAV capsid protein. The AAV capsid target protein can be a native AAV capsid protein or a mutant AAV capsid protein containing one or more mutations in the native AAV capsid protein sequence.

[0039] Target proteins for expression by use of the compositions and methods can include proteins related to enzyme replacement, such as agalsidase beta, agalsidase alpha, imiglucerase, taliglucerase alpha, velaglucerase alpha, alglucerase, sebelipase alpha, laronidase, idursulfase, elosulfase alpha, galsulfase, alglucosidase alpha, Factor VIII, C3 inhibitor, Hurler and Hunter corrective factor, etc. In some embodiments, the target protein is a biosimilar. In some embodiments, the target protein can be a secreted protein, such as C1-Inh. In some embodiments, the target protein is an antibody. In some embodiments, the compositions and methods are used for enzyme production. Such enzymes can be useful in the production of clinical test kits or other diagnostic assays. In some embodiments, the compositions and methods are used for the production of therapeutic proteins. In some embodiments, the protein is a human protein and the host cells for expression are human cells.

[0040] In some embodiments, the target protein is abarelix, abatacept, abciximab, adalimumab, aflibercept, agalsidase beta, albiglutide, aldesleukin, alefacept, alemtuzumab, alglucerase, alglucosidase alfa, alirocumab, aliskiren, alpha-1-proteinase inhibitor, alteplase, anakinra, anastrozole, anisoylated plasminogen streptokinase activator complex, anthrax immune globulin human, antihemophilic factor, antithrombin alpha, antithrombin III human, antithymocyte globulin, antithymocyte globulin (equine), antithymocyte globulin (rabbit), aprotinin, arcitumomab, asparaginase, asparaginase Erwiniachrysanthemi, atezolizumab, autologous cultured chondrocytes, basiliximab, becaplermin, belatacept, belimumab, beractant, bevacizumab, bivalirudin, blinatumomab, botulinum toxin type A, botulinum toxin type B, brentuximab vedotin, brodalumab, buserelin, C1 esterase inhibitor (human), C1 esterase inhibitor, canakinumab, canakinumab, capromab, certolizumab pegol, cetuximab, coriogonadotropin alpha, chorionic gonadotropin (human), chorionic gonadotropin, coagulation factor IX, coagulation factor VIIa, coagulation factor X human, coagulation factor XIII A subunit, collagenase, conestat alpha, corticotropin, cosyntropin, daclizumab, daptomycin, daratumumab, darbepoetin alpha, defibrotide, denileukin diftitox, denosumab, desirudin, dinutuximab, dornase alfa, drotrecogin alpha, dulaglutide, eculizumab, efalizumab, efmoroctocog alpha, erosulfase alpha, erlotinib, enfuvirtide, epoetin alpha, epoetin zeta, eptifibatide, etanercept, evolocumab, exenatide, factor IX complex (human), fibrinogen concentrate (human), fibrinolysin alias plasmin, filgrastim, filgrastim-sndz, follitropin alpha, follitropin beta, galsulfase, intrinsic factor, gemtuzumab ozogamicin, glatiramer acetate, glucagon recombinant, glucarpidase, golimumab, gramicidin D, hepatitis A vaccine, hepatitis B immune globulin, human calcitonin, human ClostridiumTetanus toxoid immunoglobulin, human rabies virus immunoglobulin, human Rho(D) immunoglobulin, human serum albumin, human varicella-zoster immunoglobulin, hyaluronidase, hyaluronidase, ibritumomab, ibritumomab tiuxetan, idarucizumab, idursulfase, imiglucerase, immunoglobulin human, infliximab, insulin aspart, bovine insulin, insulin degludec, insulin detemir, insulin glargine, insulin glulisine, insulin lispro, porcine insulin, regular insulin, regular insulin, insulin, porcine, insulin, isophane, interferon alpha-2a, recombinant, interferon alpha-2b, interferon alfacon-1, interferon alpha-n1, interferon alpha-n9, interferon beta-1a, interferon beta-1b, interferon gamma-1b, intravenous immunoglobulin, ipilimumab, ixekizumab, laronidase, lenograstim, lepirudin, leuprolide, liraglutide, lucinactant, lutropin alpha, lutropin alpha, mecasermin, menotropin, mepolizumab, epoetin beta, metreleptin, muromonab, natalizumab, alpha interferon, necitumumab, nesiritide, nivolumab, Obiltoxaximab, obinutuzumab, ocriplasmin, ofatumumab, omalizumab, oprelvekin, OspA lipoprotein, oxytocin, palifermin, palivizumab, pancrelipase, panitumumab, pembrolizumab, pertuzumab, volactant alpha, pramlintide, Preotact, protein S human, ramucirumab, ranibizumab, rasburicase, raxibacumab, reteplase, rilonacept, rituximab, romiplostim, sacrosidase, salmon calcitonin, sargramostim, satumomab pendetide (SatumomabIt is selected from the group consisting of pendetide, severinase alpha, secretin, secukinumab, sermorelin, serum albumin, iodinated serum albumin, siltuximab, simoctocog alpha, cyproislet-T, recombinant somatotropin, recombinant somatotropin, streptokinase, slodexide, susoctocog alpha, taliglucerase alpha, teduglutide, teicoplanin, tenecteplase, teriparatide, tesamorelin, thrombomodulin alpha, cymalfasin, thyroglobulin, thyrotropin alpha, thyrotropin alpha, tocilizumab, tositumomab, trastuzumab, tuberculin purified protein derivative, turoctocog alpha, urofolitropin, urokinase, ustekinumab, vasopressin, vedolizumab, and velaglucerase alpha.

[0041] In some embodiments, the target protein is, but is not limited to, a soluble protein, a secreted protein, or a membrane protein. In some embodiments, the target protein is, but is not limited to, dopamine receptor 1 (DRD1), cystic fibrosis transmembrane conductance regulator (CFTR), C1 esterase inhibitor (C1-Inh), IL2-inducible T-cell kinase (ITK), or NADase. In some embodiments, the NADase is SARM1. In some embodiments, SARM1 is a deletion variant representing the mature protein.

[0042] In some embodiments, the target protein is a membrane protein. Exemplary membrane proteins include ion channels, gap junctions, ion channel receptors, transporters, complex membrane proteins such as cell surface receptors (e.g., G protein-coupled receptors (GPCRs), tyrosine kinase receptors, integrins, etc.), proteins that shuttle between the membrane and the cytosol in response to signaling (e.g., Ras, Rac, Raf, Gα subunits, arrestin, Src, and other effector proteins), and others. In some embodiments, the membrane protein is a G protein-coupled receptor. In some embodiments, the target protein is a 7-transmembrane domain receptor, 7TM receptor, heptahelical receptor, serpentine receptor, or G protein-linked receptor (GPLR). In some embodiments, the target protein is a class A GPCR, class B GPCR, class C GPCR, class D GPCR, class E GPCR, or class F GPCR. In some embodiments, the target protein is a class 1 GPCR, class 2 GPCR, class 3 GPCR, class 4 GPCR, class 5 GPCR, or class 6 GPCR. In some embodiments, the target protein is a rhodopsin-like GPCR, secretin receptor family GPCR, metabotropic glutamate / pheromone GPCR, fungal mating pheromone receptor, cyclic AMP receptor, or Frizzled / smoothened GPCR.

[0043] In some embodiments, the target protein is a nuclease, NAD+ nuclease, hydrolase, glycosylase, glycosylase that hydrolyzes N-glycosyl compounds, NAD+ glycohydrolase, NADase, DPNase, DPN hydrolase, NAD hydrolase, diphosphopyridine nuclease, nicotinamide adenine dinucleotide nuclease, NAD glycohydrolase, NAD nuclease or nicotinamide adenine dinucleotide glycohydrolase. In some embodiments, the target protein is an enzyme involved in nicotinic acid and nicotinamide metabolism and the calcium signaling pathway.

[0044] In some embodiments, the present disclosure provides a protein expressed by introduction of the vector system (or vector) of the present disclosure into eukaryotic cells. In some embodiments, the present disclosure provides a target protein produced by a eukaryotic cell comprising the polynucleotide of the present disclosure.

[0045] Enhancer protein The present disclosure relates to the co-expression of a target protein and an enhancer protein. In some embodiments, the enhancer protein can improve one or more aspects of target protein expression, including but not limited to yield, quality, folding, post-translational modification, activity, localization and downstream activity, or reduce one or more of misfolding, altered activity, inaccurate post-translational modification and / or toxicity.

[0046] In some embodiments, the enhancer protein is a nuclear pore blocking virus protein. In some embodiments, the enhancer protein is a native or synthetic peptide that can block nuclear pores and thereby inhibit nucleocytoplasmic transport ("NCT"). In some embodiments, the enhancer protein is a virus protein. In some aspects, the virus protein is an NCT inhibitor.

[0047] In some embodiments, the enhancer protein is selected from the group consisting of picornavirus leader (L) protein, picornavirus 2A protease, rhinovirus 3C protease, coronavirus ORF6 protein, Ebola virus VP24 protein, Venezuelan equine encephalitis virus (VEEV) capsid protein, herpes simplex virus (HSV) ICP27 protein, and rhabdovirus matrix (M) protein.

[0048] The enhancer protein is a functional variant of any of the proteins disclosed herein. As used herein, the term "functional variant" refers to a protein that is homologous to the original protein and / or shares substantial sequence similarity with the original protein (e.g., greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% sequence identity) and shares one or more functional characteristics of the original protein. For example, a functional variant of an enhancer protein that is an NCT inhibitor retains the ability to inhibit NCT.

[0049] In some embodiments, the enhancer protein is a leader (L) protein derived from picornavirus or a functional variant thereof. In some embodiments, the enhancer protein is a leader protein derived from the genus Cardiovirus, Hepatovirus or Aphthovirus. For example, the enhancer protein can be derived from bovine rhinitis A virus, bovine rhinitis B virus, equine rhinitis A virus, foot-and-mouth disease virus, hepatitis A virus, hepatitis B virus, Marmota himalayana hepatitis virus, Phopivirus, cardiovirus A, cardiovirus B, Theiler's murine encephalomyelitis virus (TMEV), Vilyuisk human encephalomyelitis virus (VHEV), Theiler-like rat virus (TRV) or Saffold virus (SAF-V).

[0050] In some embodiments, the enhancer protein is the L protein of Theiler's virus or a functional variant thereof. In some embodiments, the L protein shares at least 90% identity with SEQ ID NO: 1. In some embodiments, the enhancer protein can comprise, consist of, or consist essentially of SEQ ID NO: 1. The enhancer protein can share at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with SEQ ID NO: 1.

[0051] In some embodiments, the L protein is the L protein of encephalomyocarditis virus (EMCV) or a functional variant thereof. In some embodiments, the L protein can share at least 90% identity with SEQ ID NO: 2. In some embodiments, the enhancer protein can comprise, consist of, or consist essentially of SEQ ID NO: 2. The enhancer protein can share at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with SEQ ID NO: 2.

[0052] In some embodiments, the L protein is selected from the group consisting of the L protein of poliovirus, the L protein of HRV16, the L protein of mengovirus, and the L protein of Saffold virus 2, or functional variants thereof.

[0053] In some embodiments, the enhancer protein is the picornavirus 2A protease or a functional variant thereof. In some embodiments, the enhancer protein is a 2A protease derived from enterovirus, rhinovirus, Aphtovirus or cardiovirus.

[0054] In some embodiments, the enhancer protein is a rhinovirus 3C protease or a functional variant thereof. In some embodiments, the enhancer protein is a Picornain 3C protease. In some embodiments, the enhancer protein is a 3C protease derived from an enterovirus, rhinovirus, aphthovirus, or cardiovirus. For example, in some non-limiting embodiments, the enhancer protein is a 3C protease derived from poliovirus, coxsackievirus, rhinovirus, foot-and-mouth disease virus, or hepatitis A virus.

[0055] In some embodiments, the enhancer protein is a coronavirus ORF6 protein or a functional variant thereof. In some embodiments, the enhancer protein is a viral protein that disrupts the formation of the nuclear translocation complex and / or disrupts the transport of STAT1 into the nucleus.

[0056] In some embodiments, the enhancer protein is an Ebola virus VP24 protein or a functional variant thereof. In some embodiments, the enhancer protein is an Ebola virus VP40 protein or VP35 protein. In some embodiments, the enhancer protein is a viral protein that binds to the importin protein karyopherin-α (KPNA). In some embodiments, the enhancer protein is a viral protein that inhibits the binding of STAT1 to KPNA.

[0057] In some embodiments, the enhancer protein is a Venezuelan equine encephalitis virus (VEEV) capsid protein or a functional variant thereof. In some embodiments, the enhancer protein is a viral capsid protein that interacts with the nuclear pore complex.

[0058] In some embodiments, the enhancer protein is the herpes simplex virus (HSV) ICP27 protein or a functional variant thereof. In some embodiments, the enhancer protein is the HSV ORF57 protein.

[0059] In some embodiments, the enhancer protein is the rhabdovirus matrix (M) protein or a functional variant thereof. In some embodiments, the enhancer protein is the M protein from Cytorhabdovirus, Dichorhavirus, Ephemerovirus, Lyssavirus, Novirhabdovirus, Nucleorhabdovirus, Perhabdovirus, Sigma virus, Sprivivirus, Tibrovirus, Tupavirus, Varicosavirus or Vesiculovirus.

[0060] In some embodiments, the enhancer protein is selected from the proteins listed in Table 1 or functional variants thereof. The polynucleotide encoding the enhancer protein can encode an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequences listed in Table 1. The amino acid sequence of the enhancer protein can be at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequences listed in Table 1. The amino acid sequence of the enhancer protein can be at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11. In some embodiments, the enhancer protein can have an amino acid sequence comprising, consisting of or consisting essentially of one of the amino acid sequences listed in Table 1. In some embodiments, the enhancer protein can have an amino acid sequence comprising, consisting of or consisting essentially of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.

Table 1-1

Table 1-2

Table 1-3

[0061] Fusion protein In some embodiments, the target protein and the enhancer protein are included in a single fusion protein. In some embodiments, the fusion protein can include a linker element. In some embodiments, the linker element can include a cleavage site for enzymatic cleavage. In other embodiments, the fusion protein or the linker element does not include a cleavage site, and the expressed fusion protein includes both the target protein and the enhancer protein.

[0062] Protein Modification The target protein, enhancer protein and / or fusion protein, or polynucleotide encoding such, may be modified to include one or more markers, labels or tags. For example, in some embodiments, the proteins of the present disclosure may be labeled with any label that would enable their detection, such as a radiolabel, fluorescent agent, biotin, peptide tag, enzyme fragment, or the like. The protein can include an affinity tag, such as a His-tag, FLAG-tag, GST-tag, Strep-tag, biotin-tag, immunoglobulin binding domain, such as an IgG binding domain, calmodulin binding peptide, or the like. In some embodiments, the FLAG-tag includes the amino acid sequence DYKDDDDK (SEQ ID NO: 21). In some embodiments, the polynucleotides of the present disclosure include a selectable marker, such as an antibiotic resistance marker.

[0063] Polymerase For the transcription of polynucleotides encoding a target protein(s) and an enhancer protein(s), an endogenous or exogenous polymerase can be used. In some embodiments, the transcription of the polynucleotide(s) is carried out by a natural polymerase contained in a cell (e.g., a eukaryotic cell). A viral polymerase can be used instead or in addition thereto. In some embodiments, a viral promoter is used in combination with one or more viral polymerases. In some embodiments, a eukaryotic promoter is used in combination with one or more eukaryotic polymerases. Exemplary viral polymerases include, but are not limited to, T7, T5, EMCV, HIV, influenza, SP6, CMV, T3, T1, SP01, SP2, Phi15, and others. The viral polymerase is an RNA priming or capping polymerase. In some embodiments, an IRES element is used in conjunction with the viral polymerase.

[0064] The vector(s) according to the present disclosure can include a polynucleotide sequence encoding a polymerase. In some embodiments, the polymerase is a viral polymerase. The polynucleotide sequence encoding the polymerase can be included by a vector containing a target protein-encoding polynucleotide and / or an enhancer protein-encoding polynucleotide. In some embodiments, the polymerase can be included by a vector that does not contain a target protein or enhancer protein-encoding polynucleotide.

[0065] In some embodiments, at least one of the one or more vectors contained by the systems, methods, or cells disclosed herein can include a polynucleotide sequence encoding a T7 RNA polymerase.

[0066] Vector In some embodiments, the present disclosure relates to a vector comprising nucleic acid sequences for the expression of one or more target proteins and one or more enhancer proteins. In some embodiments, the vector(s) has a first polynucleotide encoding a target protein and a second polynucleotide encoding an enhancer protein. In some embodiments, the vector(s) is any one of the expression cassettes disclosed herein, e.g., a 5' inverted terminal repeat (ITR), any one of the nucleic acid sequences disclosed herein for the expression of one or more target proteins and one or more enhancer proteins, and a 3' ITR, and / or an adeno-associated virus (AAV) expression cassette comprising a nucleic acid sequence encoding an AAV capsid protein.

[0067] Vectors for use according to the present disclosure can include any vector known in the art. In certain embodiments, the vector is any recombinant vector capable of expressing a protein or polypeptide of interest or a fragment thereof, e.g., an adeno-associated virus (AAV) vector, a lentiviral vector, a retroviral vector, a replication-competent adenoviral vector, a replication-deficient adenoviral vector, a herpesvirus vector, a baculovirus vector, or a non-viral plasmid. In some embodiments, the vector is a viral vector, plasmid, phage, phagemid, cosmid, fosmid, bacteriophage, or artificial chromosome. In some embodiments, the vector is a viral vector comprising an adenoviral vector, a retroviral vector, or an adeno-associated virus vector. In some embodiments, the vector is a bacterial artificial chromosome (BAC), plasmid, bacteriophage P1-derived vector (PAC), yeast artificial chromosome (YAC), or mammalian artificial chromosome (MAC).

[0068] The cells, systems, and methods disclosed herein can include one vector. In some embodiments, the cells, systems, and methods can include a single vector that includes a first polynucleotide encoding a target protein and a second polynucleotide encoding an enhancer protein.

[0069] The cells, systems, and methods disclosed herein can include two vectors. In some embodiments, the cells, systems, and methods can include a first vector that includes a first polynucleotide operably linked to a first promoter and a second vector that includes a second polynucleotide operably linked to a second promoter.

[0070] The cells, systems, and methods disclosed herein can include three or more vectors, and the vectors can encode target protein(s) and enhancer protein(s) in various combinations or configurations.

[0071] In some embodiments, cells are provided that include the vector(s) of the present disclosure. In some embodiments, cells are provided that include the polynucleotides of the present disclosure. In some embodiments, cells are provided that express the target protein(s) and enhancer protein(s) of the present disclosure.

[0072] Promoter The vector according to the present disclosure can include one or more promoters. The term "promoter" refers to a region or sequence located upstream or downstream of the transcription start site that is involved in the recognition and binding of RNA polymerase and other proteins for initiating transcription. The polynucleotide(s) or vector(s) according to the present disclosure can include one or more promoters. The promoter can be any promoter known in the art. The promoter can be a forward promoter or a reverse promoter. In some embodiments, the promoter is a mammalian promoter. In some embodiments, one or more promoters are native promoters. In some embodiments, one or more promoters are non-native promoters. In some embodiments, one or more promoters are non-mammalian promoters. Non-limiting examples of RNA promoters for use in the disclosed compositions and methods include U1, human elongation factor-1 alpha (EF-1 alpha), cytomegalovirus (CMV), human ubiquitin, spleen focus-forming virus (SFFV), U6, H1, tRNA Lys , tRNA Ser and tRNA Arg , CAG, PGK, TRE, UAS, UbC, SV40, T7, Sp6, lac, araBad, trp, and Ptac promoters.

[0073] The term "operably linked" as used herein refers to elements or structures in a nucleic acid sequence that are linked by an operable ability, rather than a physical arrangement. The elements or structures are capable of, or characterized by, achieving the desired operation. Those skilled in the art will recognize that elements or structures in a nucleic acid sequence need not be in tandem or adjacent order for them to be operably linked.

[0074] In some embodiments, the promoter constitutively drives the expression of one or more target proteins and / or one or more enhancer proteins; that is, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. The inducible promoter can be any inducible promoter known in the art, without limitation. In some embodiments, the expression of the inducible promoter is promoted by the presence of one or more environmental or chemical stimuli. For example, in some embodiments, the inducible promoter drives expression in the presence of chemical molecules such as tetracycline and its derivatives (such as doxycycline), cumate and its derivatives; or environmental stimuli such as heat or light.

[0075] In some embodiments, the inducible promoter is based on a tetracycline-controlled transcriptional activation system, a cumate repressor system, a lac repressor system, an arabinose-regulated pBad promoter system, an alcohol-regulated AlcA promoter system, a steroid-regulated LexA promoter system, a heat shock-inducible Hsp70 or Hsp90 promoter system, or a blue light-inducible pR promoter system. Thus, in some embodiments, the inducible promoter contains a nucleic acid sequence that binds to a tetracycline transactivator, such as a tetracycline response element. In some embodiments, the expression of the inducible promoter is turned on in the presence of tetracycline and its derivatives (Tet-On system), while in other embodiments, the expression of the inducible promoter is turned off in the presence of tetracycline and its derivatives (Tet-Off system). In some embodiments, the inducible promoter is based on a cumate repressor system. Thus, in some embodiments, the inducible promoter contains a nucleic acid sequence that binds to a CymR repressor, such as a cumate operator sequence.

[0076] In some embodiments, the expression of the inducible promoter is driven by the dimerization of a transcription factor. In some embodiments, the transcription factor is bacterial EL222, which dimerizes in the presence of blue light to drive expression from the C120 promoter or its regulatory elements. In some embodiments, the inducible promoter comprises a nucleic acid sequence derived from the C120 promoter or regulatory element.

[0077] The vectors according to the present disclosure can include one or more viral promoters that enable the transcription of one or more polynucleotides by one or more viral polymerases. In some embodiments, for example, the vector can include a T7 promoter configured for the transcription of either or both of a first polynucleotide (i.e., the target protein-encoding polynucleotide) or a second polynucleotide (i.e., the enhancer protein-encoding polynucleotide) by T7 RNA polymerase.

[0078] Expression cassette The vector(s) according to the present disclosure can include one or more expression cassettes. As used herein, the phrase "expression cassette" refers to a defined segment of a nucleic acid molecule that includes the minimal elements required for the production of another nucleic acid or protein encoded by the nucleic acid molecule. In some embodiments, the vector can include an expression cassette, and the expression cassette can include a first polynucleotide encoding a target protein and a second polynucleotide encoding an enhancer protein. In some embodiments, the expression cassette includes a first promoter operably linked to the first polynucleotide and a second promoter operably linked to the second polynucleotide. In some embodiments, the expression cassette includes a shared promoter operably linked to both the first polynucleotide and the second polynucleotide.

[0079] In some embodiments, the expression cassette comprises a coding polynucleotide comprising a first polynucleotide and a second polynucleotide linked by a polynucleotide encoding a cleavage element (e.g., a ribosome skipping site or 2A element), and the coding polynucleotide is operably linked to a shared promoter.

[0080] In some embodiments, the expression cassette comprises a coding polynucleotide encoding an enhancer protein and a target protein linked by a cleavage element (e.g., a ribosome skipping site or 2A element), and the coding polynucleotide is operably linked to a shared promoter.

[0081] In some embodiments, the expression cassette is configured for the transcription of a single messenger RNA encoding both a target protein and an enhancer protein linked by a cleavage element (e.g., a ribosome skipping site or 2A element), and the translation of the messenger RNA results in the expression of the target protein and the enhancer protein (e.g., L protein) as separate polypeptides.

[0082] In some embodiments, the expression cassette comprises a coding polynucleotide encoding an enhancer protein and a target protein as a fusion protein, with or without a polypeptide linker, and optionally, the polypeptide linker is a cleavable linker.

[0083] In some embodiments, the expression cassette is an adeno-associated virus (AAV) expression cassette comprising a 5' inverted terminal repeat (ITR), any one of the nucleic acid sequences disclosed herein for the expression of one or more target proteins and one or more enhancer proteins, and a 3' ITR. In some embodiments, the AAV expression cassette comprises a Kozak sequence, a polyadenylation sequence, and / or a stuffer sequence.

[0084] Separation element In some embodiments, the target protein(s) and enhancer protein(s) according to the present disclosure are encoded by the same vector or by separate vectors. In some embodiments, when nucleic acid sequences for one or more target proteins and one or more enhancer proteins are included by the same vector, the vector can include separation elements for separate expression of the proteins. In various embodiments, the vector is a bicistronic vector or a polycistronic vector. The separation element can be an internal ribosome entry site (IRES) or a 2A element. In some embodiments, the vector can include a nucleic acid encoding a 2A self-cleaving peptide. Exemplary 2A self-cleaving peptides include P2A, E2A, F2A, and T2A.

[0085] In some embodiments, the first polynucleotide or the second polynucleotide or both are operably linked to an internal ribosome entry site (IRES).

[0086] In some embodiments, the first polynucleotide or the second polynucleotide or both are operably linked to a 2A element.

[0087] Recombinant AAV particles The present disclosure provides a recombinant viral vector comprising any one of the expression cassettes disclosed herein. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector, a lentiviral vector, a retroviral vector, a replication-competent adenoviral vector, a replication-deficient adenoviral vector, a herpesvirus vector, or a baculovirus vector.

[0088] The present disclosure provides a method for producing a recombinant AAV (rAAV) vector, the method comprising contacting an adeno-associated virus (AAV) producer cell (e.g., a HEK293 cell) with any one of the AAV expression cassettes disclosed herein, or a vector (e.g., a plasmid or a bacmid) comprising any one of the AAV expression cassettes disclosed herein. In some embodiments, the vector (e.g., a plasmid or a bacmid) disclosed herein further comprises one or more genetic elements used in the production of AAV, such as, for example, the AAV rep and cap genes, and / or encodes a helper virus protein sequence.

[0089] In some embodiments, the method comprises contacting the AAV producer cell with one or more additional plasmids that, for example, comprise the AAV rep and cap genes, and / or encode a helper virus protein sequence. In some embodiments, the method further comprises maintaining the AAV producer cell under conditions such that AAV is produced.

[0090] The present disclosure provides an rAAV vector produced using any one of the methods disclosed herein. The produced rAAV vector can be of any serotype, such as, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV. In some embodiments, the produced recombinant AAV vector can comprise one or more amino acid modifications (e.g., substitutions and / or deletions) compared to the native AAV capsid. In some embodiments, the recombinant AAV vector is single-stranded AAV (ssAAV). In some embodiments, the recombinant AAV vector is self-complementary AAV (scAAV).

[0091] The present disclosure further provides a composition such as a pharmaceutical composition, comprising any one of the expression cassettes disclosed herein, any one of the vectors (such as any one of the recombinant AAV vectors, etc.) or any one of the AAV producer cells. In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers.

[0092] The present disclosure further provides a vaccine composition comprising any one of the expression cassettes disclosed herein, any one of the vectors (such as any one of the recombinant AAV vectors, etc.) or any one of the AAV producer cells, wherein the target protein is a protein capable of inducing an immune response against a pathogen in a subject after expression in the subject or having other therapeutic properties.

[0093] In some embodiments, the target protein is derived from a pathogen. The pathogen can be a virus, bacterium, fungus, or parasite. In some embodiments, the virus is selected from the group consisting of SARS-CoV-2, SARS-CoV-1, MERS-CoV, chikungunya virus, African swine fever virus, dengue virus, Zika virus, influenza virus (e.g., A, B, C), human immunodeficiency virus (HIV), Ebola virus, hepatitis virus (e.g., hepatitis A, B, C, D, and E), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and human papillomavirus. In some embodiments, the pathogenic parasite is Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, Entamoeba histolytica, Leishmania donovani, Trypanosoma brucei, Giardia lamblia. In some embodiments, the pathogenic bacterium is selected from the group consisting of Bacillus subtilis, Clostridium botulinum, Corynebacterium diphtheria, Enterococcus faecalis, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium leprae, Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella typhi, Staphylococcus aureus, Streptococcus pneumonia, and Vibrio cholera. In some embodiments, the vaccine composition comprises one or more adjuvants.

[0094] Transfection, transduction, transformation The terms "transfection," "transduction," and "transformation" refer to the process of introducing nucleic acid into a cell (e.g., a eukaryotic cell). The polynucleotides or vectors described herein can be introduced into a cell (e.g., a eukaryotic cell) using any method known in the art. Polynucleotides or vectors are well known in the art and can be introduced into cells by a variety of methods, some of which are selected based on the particular host cell. For example, polynucleotides can be introduced into cells using chemical, physical, biological, or viral means. Methods for introducing polynucleotides or vectors into cells include, but are not limited to, the use of calcium phosphate, dendrimers, cationic polymers, lipofection, fugene, peptide dendrimers, electroporation, cell squeezing, sonoporation, optical transfection, protoplast fusion, impalefection, hydrodynamic delivery, gene gun, magnetofection, particle gun, nucleofection, and viral transduction.

[0095] Vectors containing targeting DNA and / or nucleic acids encoding a target protein and an enhancer protein can be introduced into cells by a variety of methods (e.g., injection, transformation, transfection, direct uptake, particle gun firing, liposomes). The target protein and the enhancer protein can be expressed stably or transiently in cells using an expression vector. Expression techniques in eukaryotic cells are well known to those skilled in the art (see Current Protocols in Human Genetics: Chapter 12 ”Vector Therapy” & Chapter 13 ”Delivery Systems for Gene Therapy”).

[0096] In some embodiments, a polynucleotide or vector can be introduced into a host cell by insertion into the genome using standard methods for producing stable cell lines, such as lentiviral transfection, baculovirus gene transfer (BacMam) into mammalian cells, retroviral transfection, the use of CRISPR / Cas9 and / or transposons, as needed. In some embodiments, a polynucleotide or vector can be introduced into a host cell for transient transfection. In some embodiments, transient transfection can be effected by the use of viral vectors, helper lipids such as PEI, lipofectamine and / or Fectamine 293. Genetic elements can be encoded, for example, as DNA in a vector or as RNA derived from, for example, PCR. Genetic elements can be separated in different vectors or combined in the same vector.

[0097] Cells, cell lines, host cells Another aspect of the disclosure relates to a cell comprising a polynucleotide and / or vector encoding one or more target proteins and one or more enhancer proteins. The polynucleotide, vector, target protein and enhancer protein can be any of those described herein. The disclosure further provides a cell or cell line comprising a polynucleotide and / or vector encoding one or more enhancer proteins; these cells or cell lines may be referred to herein as "superproducer cells" or "superproducer cell lines". In some embodiments, a superproducer cell further comprises a polynucleotide and / or vector encoding one or more target proteins. Without being bound by any theory, it is believed that cells expressing one or more enhancer proteins disclosed herein can function as host cells for the expression of one or more target proteins.

[0098] In some embodiments, the cell is any eukaryotic cell or cell line. The disclosed polynucleotides, vectors, systems, and methods can be used in any eukaryotic cell line. Eukaryotic cell lines can include mammalian cell lines such as human and animal cell lines. Eukaryotic cell lines can also include insect, plant, or fungal cell lines. Non-limiting examples of such cells or cell lines generated from such cells include Bc HROC277, COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO, 5P2 / 0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), and perC6 cells, as well as insect cells such as Spodoptera fugiperda (Sf, e.g., Sf9), or fungal cells such as Saccharomyces, Pichia, and Schizosaccharomyces.

[0099] In some embodiments, the cell or cell line for expressing the target protein(s) and enhancer protein(s) is a human cell or cell line. In certain aspects, the selection of a human cell line is beneficial, for example, for post-translational modifications ("PTMs") such as glycosylation, phosphorylation, disulfide bond formation, etc. in the target protein. In some embodiments, the human cell or cell line is used for the expression of a human target protein.

[0100] In some embodiments, the cell line is a stable cell line. In some embodiments, the cell is transiently transfected with any one or more of the polynucleotides and / or vectors disclosed herein.

[0101] In some embodiments, the present disclosure provides eukaryotic cells for the expression of a target protein, the cells comprising an exogenous polynucleotide encoding an enhancer protein. In some embodiments, the exogenous polynucleotide encoding the enhancer protein is transiently transfected and / or not integrated into the genome of the cells. In some embodiments, the exogenous polynucleotide encoding the enhancer protein is stably integrated. In some embodiments, the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT). In some embodiments, the enhancer protein is selected from the group consisting of picornavirus leader (L) protein, picornavirus 2A protease, rhinovirus 3C protease, coronavirus ORF6 protein, Ebola virus VP24 protein, Venezuelan equine encephalitis virus (VEEV) capsid protein, herpes simplex virus (HSV) ICP27 protein, and rhabdovirus matrix (M) protein. The exogenous polynucleotide is operably linked to a promoter (either a native promoter or an exogenous promoter as needed). In some embodiments, the polynucleotide is operably linked to an internal ribosome entry site (IRES).

[0102] Method for protein expression The present disclosure provides a method for expressing a target protein in eukaryotic cells. The method can include introducing into the eukaryotic cells a polynucleotide encoding the target protein (a polynucleotide operably linked to a promoter). The method utilizes co-expression of an enhancer protein to enhance the expression level, solubility, and / or activity of the target protein.

[0103] In some embodiments, the expression level of a target protein expressed in combination with one or more enhancers according to the method of the present disclosure is higher than the expression level of the target protein expressed in the absence of the one or more enhancers. In some embodiments, the expression level of a target protein expressed in combination with one or more enhancers according to the method of the present disclosure is at least about 1.1-fold (e.g., about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold or about 10-fold) higher compared to the expression level of the target protein expressed in the absence of the one or more enhancers.

[0104] In some embodiments, the activity of a target protein expressed in combination with one or more enhancers according to the method of the present disclosure is higher than the activity of the target protein expressed in the absence of the one or more enhancers. In some embodiments, the activity of a target protein expressed in combination with one or more enhancers according to the method of the present disclosure is at least about 1.1-fold (e.g., about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold or about 10-fold) higher compared to the activity of the target protein expressed in the absence of the one or more enhancers.

[0105] In some embodiments, the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT). In some embodiments, the enhancer protein is selected from the group consisting of picornavirus leader (L) protein, picornavirus 2A protease, rhinovirus 3C protease, coronavirus ORF6 protein, Ebola virus VP24 protein, Venezuelan equine encephalitis virus (VEEV) capsid protein, herpes simplex virus (HSV) ICP27 protein, and rhabdovirus matrix (M) protein.

[0106] In some embodiments, the present disclosure relates to a method for producing a target protein by using a cell comprising a polynucleotide encoding one or more target proteins and one or more enhancer proteins. In some embodiments, the method is performed in a eukaryotic cell comprising one or more vectors. In some embodiments, the method is performed using the polynucleotides, vectors and cells described in the foregoing sections. In some embodiments, the vector(s) can have a first polynucleotide encoding a target protein and a second polynucleotide encoding an enhancer protein. In some embodiments, the first polynucleotide and the second polynucleotide are operably linked to one or more promoters.

[0107] There is further provided a method for recombinant expression of a target protein, the method comprising introducing into a eukaryotic cell a polynucleotide encoding a target protein operably linked to a promoter. In some embodiments, the method for expressing a target protein comprises introducing the vector system of the present disclosure into a eukaryotic cell. In some embodiments, the target protein is a membrane protein. In some embodiments, the localization of the membrane protein to the cell membrane is increased as compared to the localization observed when the membrane protein is expressed without an enhancer protein. In some embodiments, the level of a membrane-associated membrane protein expressed in combination with one or more enhancers according to the methods of the present disclosure is at least about 1.1-fold (e.g., about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold or about 10-fold) higher than the level of the membrane-associated membrane protein expressed in the absence of one or more enhancers.

[0108] In some embodiments, the expression of one or more enhancer proteins disclosed herein using the methods disclosed herein is related to, correlates with, or can result in an effect on the cell cycle of a host cell such that the number of enhancer-expressing host cells at a specific cell cycle stage is altered as compared to wild-type cells that do not express one or more enhancer proteins. In some embodiments, the expression of one or more enhancer proteins disclosed herein using the methods disclosed herein is related to, correlates with, or can result in arrest of the host cell at specific stages of the cell cycle. In some embodiments, the specific cell stage is a proliferative stage of the cell cycle, such as the G1, S, or G2 phase. In some embodiments, the expression of one or more enhancer proteins disclosed herein using the methods disclosed herein is related to, correlates with, or can result in a reduction or elimination of clonal drift in the cell.

[0109] In some embodiments, the method can include introducing into a eukaryotic cell a polynucleotide encoding an enhancer protein operably linked to a promoter. In some embodiments, the method can include transfection of a eukaryotic cell with one or more DNA molecules, transduction of a eukaryotic cell with a single viral vector, and / or transduction of a eukaryotic cell with two or more viral vectors.

[0110] Downstream applications In some embodiments, the target proteins produced by the use of the compositions, systems, and methods herein and the cells expressing such proteins are isolated, purified, and / or used for downstream applications. Exemplary applications include, but are not limited to, small molecule screening, structure determination (e.g., X-ray crystallography, cryo-electron microscopy, etc.), activity assays, therapeutics, enzyme replacement therapy, screening assays, diagnostic assays, clinical test kits, drug discovery, antibody discovery, and others. In some embodiments, the compositions and methods herein are used for the production of antibodies or antigens for antibody screening assays. In some embodiments, the cells expressing the target protein can be used as an assay system for screening cell interactions, antibody binding, or small molecule effects, for example, in a whole cell system.

[0111] In some embodiments, the present disclosure provides systems and methods for antibody discovery. In some embodiments, the present disclosure provides a method for generating an antibody against a target protein, the method comprising immunizing a subject with a cell or a target protein produced using the system or method of the present disclosure. In various embodiments, the subject to be immunized is a mouse, rat, rabbit, non-human primate, llama, camel, or human. Cells isolated from the subject can be subjected to further rounds of selection as isolated cells or, optionally, after generating hybridomas from the isolated cells. Polynucleotide sequences (s) encoding the heavy and light chains can be isolated from the isolated cells or hybridomas using gene cloning and / or sequencing. Gene cloning and / or sequencing can be applied to a single cell or a population of cells. In some embodiments, the compositions and methods of the present disclosure are used for the immunization of a subject and the production of polyclonal antibodies by subsequent collection of serum from the subject.

[0112] The present disclosure provides a method for antibody discovery by cell sorting, comprising the steps of preparing a solution comprising a labeled cell or target protein produced using the system or method of the present disclosure, and a population of recombinant cells, wherein the recombinant cells express a library of polypeptides each comprising an antibody or an antigen-binding fragment thereof; and sorting one or more recombinant cells from the solution by detecting recombinant cells bound to the labeled cell or labeled target protein. In other variations, cell sorting is performed on cells derived from an immunized subject. The subject can be immunized with a cell or target protein produced according to the method of the present disclosure or with another suitable immunogen. In some embodiments, the recombinant cells comprise a naive antibody library, optionally a human naive antibody library. Various antibody library generation methods are known in the art and can be combined with the method of the present disclosure. As used herein, the terms "sorting" or "cell sorting" refer to fluorescence-activated cell sorting, magnetic-activated cell sorting, and other means of selecting labeled cells in a population of labeled and unlabeled cells.

[0113] The present disclosure further provides a method for panning a phage display library, comprising the steps of mixing the phage display library with a cell or target protein produced using the system or method of the present disclosure; and purifying and / or enriching members of the phage display library that bind to the cell or target protein. In some embodiments, the phage display library expresses a population of single-chain variable fragments (scFvs) or other types of antibodies / antibody fragments (such as Fab).

[0114] In further embodiments, the present disclosure provides a method for screening for any type of protein binder. Using the cells and target proteins of the present disclosure, libraries of various types of molecules, including drugs and macromolecules (proteins, nucleic acids, and protein:nucleic acid complexes), can be screened to identify binding partners of the target protein. In other embodiments, the systems and methods of the present disclosure are used for the expression of libraries of target proteins in a single well, a pool of several sequences, or a library of gene sequences.

[0115] The ability to express the antigen in its native or disease-associated form, in high yield and / or on the surface of the cell, enables more reliable discovery and / or generation of antibodies, antibody fragments, and other molecules than prior art methods. Such antibodies, antibody fragments, and other molecules may be useful as therapeutic agents and / or research tools, or for other applications.

[0116] In some embodiments, the systems and methods of the present disclosure are suitable for use in the discovery of antibodies that bind to and / or are specific for a particular glycosylation pattern in a target molecule (e.g., a glycoprotein). In some embodiments, antibody libraries are selected against native glycosylated proteins and counterselected against inappropriately glycosylated or deglycosylated cognate proteins. Similarly stated, antibodies can be specifically selected against glycosylation patterns by using deglycosylating enzymes. In further embodiments, the cells and / or target proteins of the present disclosure are used to confirm the binding and / or functional activity of novel antibodies or other macromolecules.

[0117] In some embodiments, the systems and methods of the present disclosure are suitable for use in the biosynthesis of any target protein in any host cell disclosed herein or known in the art. For example, the systems and methods of the present disclosure are suitable for use in the biosynthesis of any target protein using fermentation in mammalian cells or in bacteria, yeast, and other microorganisms. In some embodiments, the systems and methods of the present disclosure are suitable for use in the biosynthesis of non-protein molecules by introduction of specific metabolic pathways into host cells. For example, the non-protein molecule is an opioid molecule or another metabolite.

[0118] Exemplary advantages The compositions, systems, and methods can have a number of advantages. For example, as demonstrated in Example 11, human NADase, which normally causes apoptosis and thus produces undetectable yields when overexpressed in human cell lines, can be reliably expressed and produce yields greater than 20 mg / L when an enhancer protein is co-expressed with this target protein. Moreover, the NADase expressed by this exemplary method is functional (as demonstrated by the phosphate release assay) and exhibits low between-batch variability.

[0119] Similarly, in some embodiments, the methods, systems, and cells are used for the reliable expression of proteins that are difficult to express. In some embodiments, the present disclosure relates to the production of proteins having low between-batch variability. Proteins produced in accordance with the present disclosure can exhibit one or more of the following improvements: purification without the use of a purification tag fusion; improved functional activity; reliable production; consistent activity; and suitability for therapeutic applications.

[0120] The cells of the present disclosure can have one or more of the following advantages from the perspective of target protein expression: higher concentration of target membrane protein in the membrane; slower / decreased target protein degradation; improved signal-to-noise ratio in the lysate; target protein and / or enhancer protein expression that does not affect downstream cell metabolism; increased stability against desensitization of membrane-bound membrane proteins; and higher target protein yield. Example 1 provides an exemplary example of the expression of an enhancer protein that does not affect the downstream metabolism of cells. The GPCR exemplified in Example 1 was able to interact with its natural substrate and produce activation that could be measured in vitro.

[0121] In some embodiments, the present system and method can have one or more of the following advantages: compatibility with any eukaryotic cell type; reduced need for target protein expression optimization; and reliable expression of proteins that are difficult to express.

[0122] System One aspect of the present disclosure provides a system for recombinant expression of a target protein in a eukaryotic cell comprising one or more vectors. The vector(s) can have a first polynucleotide encoding a target protein and a second polynucleotide encoding an enhancer protein. The enhancer protein can be an inhibitor of nucleocytoplasmic transport (NCT). In some embodiments, the enhancer protein can be selected from the group consisting of picornavirus leader (L) protein, picornavirus 2A protease, rhinovirus 3C protease, herpes simplex virus (HSV) ICP27 protein, and rabies virus matrix (M) protein. The first polynucleotide and the second polynucleotide can be operably linked to one or more promoters.

[0123] In some embodiments, the enhancer protein is an inhibitor of nuclear cytoplasmic transport (NCT). In some embodiments, the NCT inhibitor is a viral protein.

[0124] In some embodiments, the enhancer protein is an NCT inhibitor selected from the group consisting of picornavirus leader (L) protein, picornavirus 2A protease, rhinovirus 3C protease, coronavirus ORF6 protein, Ebola virus VP24 protein, Venezuelan equine encephalitis virus (VEEV) capsid protein, herpes simplex virus (HSV) ICP27 protein, and rhabdovirus matrix (M) protein.

[0125] The NCT inhibitor can be a picornavirus leader (L) protein or a functional variant thereof. In some embodiments, the NCT inhibitor can be a picornavirus 2A protease or a functional variant thereof. In some embodiments, the NCT inhibitor can be a rhinovirus 3C protease or a functional variant thereof. In some embodiments, the NCT inhibitor can be a coronavirus ORF6 protein or a functional variant thereof. In some embodiments, the NCT inhibitor can be an Ebola virus VP24 protein or a functional variant thereof. In some embodiments, the NCT inhibitor can be a Venezuelan equine encephalitis virus (VEEV) capsid protein or a functional variant thereof. In some embodiments, the NCT inhibitor is a herpes simplex virus (HSV) ICP27 protein or a functional variant thereof. In some embodiments, the NCT inhibitor is a rhabdovirus matrix (M) protein or a functional variant thereof.

[0126] In some embodiments, the enhancer protein is an L protein that is the L protein of Theiler virus or a functional variant thereof. In some embodiments, the L protein can share at least 90% identity with SEQ ID NO: 1.

[0127] In some embodiments, the L protein is the L protein of encephalomyocarditis virus (EMCV) or a functional variant thereof. In some embodiments, the L protein can share at least 90% identity with SEQ ID NO: 2.

[0128] In some embodiments, the L protein is selected from the group consisting of the L protein of poliovirus, the L protein of HRV16, the L protein of mengovirus, and the L protein of sapovirus 2, or functional variants thereof.

[0129] The system can include a single vector containing an expression cassette, and the expression cassette includes a first polynucleotide and a second polynucleotide. In some embodiments, the expression cassette includes a first promoter operably linked to the first polynucleotide and a second promoter operably linked to the second polynucleotide. In some embodiments, the expression cassette includes a shared promoter operably linked to both the first polynucleotide and the second polynucleotide.

[0130] In some embodiments, the expression cassette includes a coding polynucleotide that includes a first polynucleotide and a second polynucleotide linked by a polynucleotide encoding a ribosome skipping site, and the coding polynucleotide is operably linked to a shared promoter.

[0131] In some embodiments, the expression cassette includes a coding polynucleotide, and the coding polynucleotides encoding the enhancer protein and the target protein are linked by a ribosome skipping site, and the coding polynucleotide is operably linked to a shared promoter.

[0132] In some embodiments, the expression cassette is configured for the transcription of a single messenger RNA encoding both a target protein and an enhancer protein (e.g., L protein) linked by a ribosome skipping site; the translation of the messenger RNA results in the expression of the target protein and the enhancer protein (e.g., L protein) as separate polypeptides.

[0133] The system can include one type of vector. In some embodiments, the system can include a single vector containing a first polynucleotide encoding a target protein and a second polynucleotide encoding an enhancer protein.

[0134] The system can include two types of vectors. In some embodiments, the system can include a first vector containing a first polynucleotide operably linked to a first promoter and a second vector containing a second polynucleotide operably linked to a second promoter.

[0135] In some embodiments, the first polynucleotide or the second polynucleotide or both are operably linked to an internal ribosome entry site (IRES).

[0136] In some embodiments, at least one of the one or more vectors included by the system can include a T7 promoter configured for the transcription of either or both of the first polynucleotide and the second polynucleotide by T7 RNA polymerase.

[0137] In some embodiments, at least one of the one or more vectors included by the system can include a polynucleotide sequence encoding T7 RNA polymerase.

[0138] All papers, publications, and patents cited in this specification are hereby incorporated by reference as if each individual paper, publication, or patent was specifically and individually indicated as being incorporated herein by reference, and are incorporated by reference to disclose and describe methods and / or materials in connection with those publications that are cited. However, any reference to any reference, article, publication, patent, patent publication, and patent application cited herein is not an admission or any form of suggestion that such constitutes valid prior art or forms part of the common general knowledge in any country in the world, and should not be taken as such.

[0139] It is specifically intended that, unless the context indicates otherwise, the various features described herein can be used in any combination.

[0140] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

Examples

[0141]

Table 4

[0142] The structural genes used for plasmid construction were synthesized by phosphoramidite chemistry, amplified, and cloned into the vectors described above using isothermal assembly reactions such as NEB HI-FI or Gibson assembly with the primers listed in Table 2. The selected amino acid sequences included in the exemplary constructs used in these examples are presented in Table 3.

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 3-1

Table 3-2

Table 3-3

Table 3-4

[0143] Cell line - Culture and transfection The application of the present system, method and composition in human eukaryotic cells was demonstrated using HEK293 cells. HEK293 adherent cells (CLS) were cultured in Dulbecco's Modified Eagle Medium high glucose (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 50,000 U Pen Strep (Gibco). Before transient transfection using 293 Fectin (ThermoFisher) according to the manufacturer's instructions, HEK293 cells were grown at 37 °C and 5% CO 2 until they reached a confluency of 80%. The cells were detached using 0.5% trypsin solution at 37 °C for 5 minutes, and protein-expressing cells were collected 48 hours later by scraping. The cells were pelleted (5,000×g, 15 minutes, 4 °C), and the supernatant was discarded. The cell pellet was stored at -80 °C until further use.

[0144] The application of the present system, method and composition in human eukaryotic cells was demonstrated using suspension HEK293 cells. Suspension-adapted HEK293 cells (CLS) were cultured in supplemented Expi293 Expression Medium (Gibco). One day before transfection, cells were seeded at 1.75×10 6 cells / ml and incubated overnight at 37 °C and 5% CO 2 and then transiently transfected using the Expi293 Expression System Kit (Gibco) according to the manufacturer's instructions. Protein-expressing cells were collected by centrifugation (5,000×g, 15 minutes, 4 °C) 48 - 96 hours later. For soluble or membrane proteins, the supernatant was discarded, and the cell pellet was stored at -80 °C until further use. For secreted proteins, the supernatant was used immediately for further purification.

[0145] The application of the present system, method, and composition in eukaryotic animal cells was demonstrated using CHO-K1 cells. CHO-K1 adherent cells (CLS) were cultured in DMEM / F-12 GlutaMAX medium (Gibco) supplemented with 10% fetal bovine serum (Gibco). Before transient transfection using Lipofectamine LTX (ThermoFisher) according to the manufacturer's instructions, CHO-K1 cells were grown at 37 °C and 5% CO2 until they reached 80% confluence. Protein-expressing cells were collected 48 hours later by detaching the cells using a 0.5% trypsin solution for 5 minutes at 37 °C and scraping. The cells were pelleted (5,000×g, 15 minutes, 4 °C), and the supernatant was discarded. The cell pellet was stored at -80 °C until further use.

[0146] The application of the present system, method, and composition in eukaryotic insect cells was demonstrated using SF9 cells. SF9 suspension cells (CLS) were cultured in Sf9-900 III medium (Gibco). Before seeding in 6-well plates for transient transfection using Cellfectin II (ThermoFisher) according to the manufacturer's instructions, SF9 cells were grown at 26 °C and 130 rpm. Protein-expressing cells were collected 48 hours later by detaching and pelleting (5,000×g, 15 minutes, 4 °C), and the supernatant was discarded. The cell pellet was stored at -80 °C until further use.

[0147] (Example 1) GFP Expression in HEK293 Cells CMV Promoter System To demonstrate the effect of introducing viral nuclear pore blocking proteins during expression, HEK293 cells were transfected with either EG1 or EG2, or co-transfected with EG3 and EG4 constructs (see Table 2 and Figure 2 for construct details). Expression of the viral pore blocking protein resulted in a controlled regulation of protein expression. Consequently, the resulting GFP signal decreased. The reason for the controlled regulation of the gene of interest in cooperation with the pore blocking protein is the mechanism of action of the viral protein. Without being limited by theory, a possible mechanism of protein regulation is that by expressing the pore blocking protein, nuclear export of mRNA can be inhibited, and as a result, translation of the target protein will be downregulated. After stabilization, the pore blocking protein will be degraded and mRNA transport will resume. This will again result in the expression of both the target protein and enhancer proteins, such as the pore blocking protein. This tightly controlled feedback ensures the stabilization and persistent expression of the target protein and prevents the normal regulation of eukaryotic cells that leads to the cessation of protein expression.

[0148] Figures 3A - 3D show the effect on GFP expression in the absence and presence of the L protein from ECMV as an exemplary enhancer protein according to the present disclosure. Prior to transiently transfecting either EG1 or EG2 as described above, HEK293 cells were seeded in 24-well plates at 0.05 × 10 6 cells / well and incubated overnight at 37°C and 5% CO 2 . GFP expression was monitored at 24 hours and 48 hours using a fluorescence microscope. Images were taken using a CCD camera (Amscope) and analyzed with ISCapture (Amscope). This example demonstrates the improved regulation of target protein expression in an exemplary system comprising a target protein polynucleotide and an enhancer protein polynucleotide according to the present disclosure.

[0149] T7 polymerase system EG2 uses the native polymerase of a eukaryotic host, but can initiate transcription outside the nucleus using other viral polymerases such as T7. The viral polymerase is under the control of a standard eukaryotic promoter, and the corresponding mRNA will likely rely on nuclear export. In the cytosol, the viral polymerase is translated and then initiates transcription of the target protein polynucleotide and the enhancer protein polynucleotide. In some embodiments, as a result of the expression of the enhancer protein, nuclear transport of the viral polymerase will be reduced. Stabilization of the system will result in degradation of the enhancer protein and resumption of mRNA transport of the viral polymerase. Without being bound by theory, this feedback can prevent normal regulation of the cell while overexpressing the recombinant protein. In some situations, the use of a viral polymerase provides the advantage of higher expression levels on a cell-to-cell basis compared to systems using eukaryotic polymerases.

[0150] Figures 4A - 4D show the successful expression of GFP in cooperation with the L protein derived from ECMV from the T7 promoter when co-transfected with a vector having T7. Prior to transiently transfecting either EG1 or EG3 and EG4 as described above, HEK293 cells were seeded at 0.05×10 6 cells / well in a 24-well plate and incubated overnight at 37 °C and 5% CO 2 . GFP expression was monitored at 24 and 48 hours using a fluorescence microscope. Images were taken using a CCD camera (Amscope) and analyzed with ISCapture (Amscope). This example demonstrates the successful use of T7 as an exemplary viral polymerase in cooperation with GFP as the target protein and the L protein of ECMV as the enhancer protein. Similar to the example above, the introduction of the L protein resulted in more stringent regulation of expression and thus an overall reduction in overexpression.

[0151] (Example 2) Production of Dopamine Receptor 1 (DRD1) To obtain a high-density active membrane receptor, the application of the disclosed systems and methods for the co-expression of a membrane protein as a target protein in combination with a pore-blocking protein as an enhancer protein using DRD1 was described. DRD1 is a G protein-coupled receptor and is known to be difficult to express using academic standards. In this system, a DRD1-GFP fusion (EG8) was used to visualize the accurate translocation to the outer membrane of the cell. An academic standard (EG10) was used as a control to illustrate the problems associated with GPCRs in academic and industrial settings.

[0152] Improved Membrane Protein Expression and Membrane Localization The DRD1-GFP fusion was expressed in HEK293 cells. Before transiently transfecting either EG10 or EG8 as described above, HEK293 cells were seeded at 0.05×10 6 cells / well in a 24-well plate and incubated overnight at 37 °C and 5% CO 2 . DRD1-GFP expression was monitored at 24 and 48 hours using a fluorescence microscope. Images were taken using a CCD camera (Amscope) and analyzed with ISCapture (Amscope).

[0153] Figures 5A - 5D demonstrate that EG10 is unable to accurately translocate the expressed receptor. Without being bound by theory, as a result of overexpression of the human DRD1 receptor in human cells having the EG10 construct, the cells appear to initiate degradation or control of the expressed target protein. This form of regulation results in the formation of misfolded proteins as inclusion bodies (Figure 5B, red arrow). The regulation of membrane protein expression by cells in this manner can result in inactive and misfolded proteins and, consequently, expressed proteins of unusable and insufficient quality. In contrast, co-expression of the target membrane protein and the exemplary enhancer protein, as evidenced by accurate insertion into the membrane and the absence of inclusion bodies, resulted in accurately translocated DRD1 - GFP (Figures 5C - 5D). This example demonstrates that co-expression of an exemplary enhancer protein (the L protein of ECMV) along with an exemplary target membrane protein (DRD1) resulted in improved expression and localization of the membrane protein. Without being bound by theory, this system appears to bypass the normal regulation of the cell that would result in degradation of the expressed membrane protein, resulting in strict regulation of target protein expression. Thus, this system is suitable for high - yield expression and purification of GPCRs.

[0154] Expression of target and enhancer proteins from different constructs To illustrate that the enhancer protein can be encoded by a separate DNA molecule, the DRD1 - GFP (EG10) construct was co - expressed with the L protein from ECMV (EG11) under the control of separate promoters in separate vectors. Prior to transiently transfecting EG10 and EG11 as described above, HEK293 cells were seeded at 0.05×10 6 cells / well in a 24 - well plate and incubated overnight at 37°C and 5% CO 2 2. DRD1 - GFP expression was monitored after 48 hours using a fluorescence microscope. Images were taken and analyzed by an Echo Revolve microscope system.

[0155] Figures 10A and 10B demonstrate that co-expression of L-protein and DRD1-GFP from two separate vectors ensures accurate membrane association. Expression of DRD1-GFP results in inclusion body formation (Figure 10A, red arrow), but accurate membrane association can be achieved by co-expression of the L-protein. Figure 10B demonstrates that the regulatory effect of the L-protein is sufficient to restore accurate membrane association of DRD1 even when the L-protein is expressed from separate vectors and promoters.

[0156] These results demonstrate that using the methods disclosed herein, the enhancer proteins and target proteins disclosed herein can be expressed from separate constructs to achieve an improvement in the yield and / or functionality of the expressed target protein. Furthermore, these results suggest that expression of any target protein from any construct or vector currently known or used in the art can be combined with the expression of one or more of the enhancer proteins disclosed herein from the same construct or a different construct to improve the yield and / or functionality of the expressed target protein. This dramatically enhances the versatility of the methods and compositions disclosed herein.

[0157] Functional Activity of Membrane Proteins In addition to the description of the correctly translocated GPCRs such as DRD1, an activity assay was performed using the DRD1-Strep fusion. The smaller strep-tag ensures that the interaction with the cytosolic located G protein is intact and that a functional assay can be performed. After dopamine binding, DRD1 releases the heterotrimeric G protein into its Gα subunit and its Gβγ complex. In the resting state, Gα binds to GDP, but after activation, it exchanges GDP for GTP. The Gα-GTP complex interacts with adenylate cyclase (AC), resulting in the activation of AC activity and consequently an increase in cAMP levels. Changes in intracellular cAMP levels can be measured by a standard cAMP assay. Academic and industrial standards (EG5) were compared to the same target protein in co-expression with the L protein of ECMV.

[0158] The DRD1-Strep fusion was expressed in HEK293 cells. HEK293 cells were seeded at 5,000 cells / well in 96-well white clear-bottom plates and incubated overnight at 37 °C and 5% CO 2 before transiently transfecting either EG5 or EG6 as described above. Proteins were expressed for 48 h and DRD1 activity was analyzed using the cAMP-Glo™ assay (Promega) according to the manufacturer's instructions. After 48 h, cells were washed with sterile PBS pH 7.2 and incubated with 20 μl of a 1 mM dopamine substrate solution (+ dopamine; on) or PBS pH 7.2 (- dopamine; off) for 2 h at 37 °C. After incubation, cells were washed with PBS pH 7.2 and subsequently 20 μl of lysis buffer was added. Lysis was performed for 15 min at room temperature (RT) with shaking. Then, 40 μl of detection solution was added and cells were incubated for 20 min at RT with shaking. Prior to analysis, the reaction was stopped using 80 μl of Kinase-Glo® reagent incubated for 15 min at RT. Luminescence was measured using a plate reader (BioTek Synergy™ LX) and data were analyzed using a standard analysis program.

[0159] Figure 11 demonstrates the advantage of expressing DRD1-Strep in cooperation with the L protein derived from EMCV. When dopamine is added to cells expressing DRD1, the corresponding luminescence signal drops as a result of internal cAMP release. Figure 11 shows that co-expression of DRD1 and the L protein derived from EMCV results in a strong activation signal, as indicated by the difference between the off state in the absence of dopamine and the on state in the presence of dopamine. An important aspect of the assay is to exclude false activation of DRD1 or cAMP release in the absence of the activator, dopamine. If the assay produces a "leaky" signal, its usefulness for drug discovery screening is low. Figure 11 shows that co-expression of DRD1 and the L protein derived from EMCV significantly reduces "leaky" activation and thus false-negative readouts when comparing the simple off signal to non-transfected cells. Therefore, co-expression of enhancer proteins using the methods disclosed herein results in more stringent regulation of the activation of the target DRD1 protein. Therefore, the methods disclosed herein have applicability in drug discovery screening.

[0160] (Example 3) Expression of DRD1-GFP using a viral promoter combined with a viral polymerase For this example, the T7 promoter was used to express DRD1-GFP as an exemplary target membrane protein that is difficult to express, demonstrating that transcription can be initiated extranuclearly using a viral polymerase such as T7. Similar to Example 1, the viral polymerase was placed under the control of a standard eukaryotic promoter, and the corresponding mRNA relied on nuclear export.

[0161] Figures 6A - 6B demonstrate the successful expression of DRD1 - GFP in cooperation with the L protein derived from ECMV from the T7 promoter when co - transfected with a vector having T7. Before transiently transfecting either EG10 or EG12 and EG4, HEK293 cells were seeded in 24 - well plates at 0.05×10 6 cells / well and incubated overnight at 37°C and 5% CO 2 . DRD1 - GFP expression was monitored at 24 hours and 48 hours using a fluorescence microscope. Images were taken using a CCD camera (Amscope) and analyzed with ISCapture (Amscope). This example demonstrates the successful use of T7 as a viral polymerase in cooperation with DRD1 - GFP as a target protein and the L protein of ECMV as an enhancer protein.

[0162] (Example 4) Expression of DRD1 - GFP Using Different Mammalian Promoters The systems, methods, and compositions according to the present disclosure are compatible with a wide variety of mammalian promoters. To demonstrate the compatibility of the co - expression of target proteins and enhancer proteins from different promoters, DRD1 - GFP was used as an exemplary target protein. As described in Example 2, the accurate expression and translocation of DRD1 - GFP can be easily detected by a fluorescence microscope. The constructs used in the experiment were engineered to express DRD1 from either the CMV promoter (EG8), the EF1 - α promoter (EG22), or the SV40 promoter (EG23), and also to have the following elements - a nucleic acid sequence encoding DRD1 - GFP, a nucleic acid sequence encoding IRES, and a nucleic acid sequence encoding the L protein sequence. An academic standard system (EG10) was used to explain the difference between accurate and inaccurate membrane association.

[0163] DRD1-GFP fusions under the control of different mammalian promoters were expressed in HEK293 cells. Before transiently transfecting any of EG8, EG10, EG22, or EG23 as described above, HEK293 cells were seeded at 0.05×10 6 cells / well in 24-well plates and incubated overnight at 37 °C and 5% CO 2 . DRD1-GFP expression was monitored 48 hours later using a fluorescence microscope. Images were taken and analyzed by an Echo Revolve microscope system.

[0164] Figure 12 demonstrates that different promoters can be used to drive target protein expression in combination with enhancer protein expression. Expression of DRD1-GFP from the control construct shows that DRD1 cannot localize to the outer membrane of the cell but rather localizes to inclusion bodies (bright green spots, Figure 12A), while all of the DRD1-GFP expressed in combination with the L-protein enhancer expressed from the CMV, EF1α, and SV40 (Figures 12B–12D) promoters accurately associate with the membrane as judged by the absence of inclusion bodies. As expected, different promoters result in different expression levels, and thus the amount of DRD1-GFP in the membrane (total fluorescence) varies.

[0165] (Example 5) Expression of DRD1-GFP using different viral pore-blocking proteins The exemplary target fusion protein DRD1-GFP was expressed in HEK293 cells in combination with different enhancer proteins. The constructs used in this experiment encoded DRD1-GFP and one of the enhancer proteins selected from the leader protein of ECMV (EG8), the leader protein of Theiler's virus (EG19), the 2A protease of poliovirus (EG21), and the M protein of vesicular stomatitis virus (EG20). As described in Example 2, the accurate expression and translocation of DRD1-GFP can be easily detected by fluorescence microscopy. The academic standard system (EG10) was used to explain the difference between accurate and inaccurate membrane association. Before being transiently transfected with any of EG8, EG10, EG19, EG20, or EG21 as described above, HEK293 cells were seeded at 0.05×10 6 cells / well in a 24-well plate and incubated overnight at 37°C and 5% CO 2 . DRD1-GFP expression was monitored 48 hours later using a fluorescence microscope. Images were taken and analyzed by an Echo Revolve microscope system.

[0166] Figure 13 demonstrates that the leader protein of ECMV (Figure 13B), the leader protein of Theiler's virus (Figure 13C), the 2A protease of poliovirus (Figure 13D), and the M protein of vesicular stomatitis virus (Figure 13E) are all sufficient to ensure accurate membrane uptake of DRD1-GFP, in contrast to DRD1-GFP (Figure 13A) which does not have any enhancer proteins.

[0167] These results indicate that several different viral pore-blocking proteins share the ability to improve the yield, localization, and / or functionality of a target protein when expressed together with the target protein in a host cell. Without being bound by theory, it is believed that the blockade of nuclear pores resulting from the expression of any one of these enhancer proteins may bypass the normal regulation of the cell that would have led to the degradation of the expressed target protein. Thus, this common mechanism by which viral pore-blocking proteins enhance target protein expression, localization, and activity enables the methods disclosed herein to be carried out by any pore-blocking protein known in the art, discovered in the future, or disclosed herein.

[0168] (Example 6) Expression of DRD1-GFP in CHO cells The experiment of Example 2 was repeated using CHO-K1 (Chinese hamster ovary) cells instead of HEK293 cells. DRD1-GFP was expressed from the EG19 construct that also encodes an enhancer protein or from the control EG10 construct.

[0169] The DRD1-GFP fusion protein was expressed in CHO-K1 cells. Prior to transiently transfecting either EG10 or EG19 using Lipofectamine 3000 (Thermofisher) according to the manufacturer's instructions, CHO-K1 cells were seeded at 0.05×10 6 cells / well in a 24-well plate and incubated overnight at 37°C and 5% CO 2 . DRD1-GFP expression was monitored 48 hours later using a fluorescence microscope. Images were taken and analyzed by an Echo Revolve microscope system.

[0170] Figure 14 demonstrates that EG10 is unable to accurately translocate the expressed receptor. Interestingly, the results of overexpression of the human DRD1 receptor in CHO cells appear to be more severe compared to HEK cells. With the EG10 construct, the cells initiate degradation or control of the expressed target protein, leading to the formation of denatured proteins as inclusion bodies (Figure 14A, red arrow). The control of membrane protein expression by cells in this manner can result in inactive and misfolded proteins and, consequently, unusable and poorly quality expressed proteins. In contrast, co-expression of the target membrane protein and the exemplary enhancer protein resulted in accurately translocated DRD1-GFP, as evidenced by the accurate insertion into the membrane and the absence of inclusion bodies (Figure 14B). This example demonstrates that co-expression of an exemplary enhancer protein (the L protein of the Taylor virus) together with an exemplary target membrane protein (DRD1) results in improved expression and localization of the membrane protein. Moreover, this example demonstrates that various eukaryotic cell types (e.g., HEK293 or CHO cells) can be used in the practice of the disclosed method.

[0171] (Example 7) Production of expression of DRD1-GFP in Sf9 cells The experiment of Example 2 was repeated using Sf9 (Spodoptera frugiperda) cells instead of HEK293. DRD1-GFP was expressed from the EG8 construct or the industrial and academic standard construct, EG10.

[0172] The DRD1-GFP fusion was expressed in Sf9 cells. Sf9 cells were seeded at 0.4×10 in a 6-well plate prior to transiently transfecting either EG10 or EG8 using Cellfectin Reagent II (Thermofisher) according to the manufacturer's instructions. 6Cells were seeded at [[ID=]], incubated at RT for 15 minutes. DRD1-GFP expression was monitored after 72 hours using a fluorescence microscope. Images were taken and analyzed by an Echo Revolve microscope system.

[0173] Figure 15 demonstrates that not only can EG10 not accurately translocate the expressed receptor, but also the expressed receptor is highly toxic to the cells. The highest fluorescence signal was observed in cells that died as a result of the toxicity of the expressed gene (Figure 15A, red arrow). In contrast, the expression of DRD1-GFP using the disclosed method prevents the cytotoxicity caused by the expression of DRD1-GFP, and receptors incorporated into the membrane are observed (Figure 15B, red arrow). Interestingly, the results of overexpression of the human DRD1 receptor in Sf9 cells appear to be more severe compared to HEK cells. Unregulated expression, such as that of the standard system EG10, causes high cell death and consequently unusable proteins. The toxic effects are dramatically milder when expressing DRD1-GFP and the L protein from EG8, as is evident from the overall cell health and membrane-bound receptors. This example demonstrates that the co-expression of an exemplary enhancer protein (the L protein of EMCV) together with an exemplary target membrane protein (DRD1) resulted in improved expression and localization of the membrane protein, along with a clearly improved control of the toxic effects. Moreover, this example demonstrates that the disclosed method is compatible with various eukaryotic cell types.

[0174] (Example 8) Production of interleukin-2-inducible T-cell kinase (ITK) Using ITK as an exemplary target protein, the application of the disclosed system for expressing soluble proteins that are typically difficult to express was illustrated. ITK is a member of the TEC family of kinases and is thought to play a role in T cell proliferation and differentiation in T cells. Also, ITK was used to demonstrate the consistency of enzyme activity between batches and the scalability of the methods disclosed herein. ITK was expressed in 3 x 10 ml, 100 ml, and 1000 ml of growth medium. Additionally, using the ITK-L-his protein fusion construct (EG9), it was demonstrated that enhancer proteins can be fused to recombinantly expressed target proteins without losing the ability to control regulation. ITK-his fusions were expressed from EG17, as well as from academic and industrial standards (EG18) as a comparison.

[0175] ITK-his and ITK-L-his fusions were expressed in HEK293 cells. Prior to transiently transfecting any of EG9, EG17, or EG18 as described above, HEK293 cells were seeded at 2 x 10 6 cells / ml in 10 ml, 100 ml, or 1000 ml of Expi293 medium and incubated overnight at 37 °C, 120 rpm, and 5% CO 2 . Cells were harvested after 48 hours (5,000 x g, 15 minutes, 4 °C), and the cell pellet was stored at -80 °C until further use.

[0176] To purify ITK, cells were lysed in lysis buffer (40 mM Tris, 7.5; 20 mM MgCl 2 ; 0.1 mg / ml BSA; 50 μM DTT; and 2 mM MnCl 2, resuspended in protease inhibitor, DNAse), lysed by sonication (2 minutes, 10 seconds on, 10 seconds off, 40% amplitude), and the crude cell extract was clarified (5,000×g, 20 minutes, 4°C). Before loading the clarified lysate using a peristaltic pump, a 5 ml His-resin column (GE Healthcare HisTrap) was equilibrated with wash buffer (40 mM Tris, 7.5; 20 mM MgCl2; 0.1 mg / ml BSA; 50 μM DTT; and 2 mM MnCl2). After loading, purification was performed in an AKTA™ system (Cytiva Life Sciences (formerly GE Healthcare)). The column was washed with 5 CV of wash buffer before eluting with a continuous gradient of 0 - 100% elution buffer (wash buffer + 300 mM imidazole) over 25 CV. Protein-containing fractions were analyzed by SDS-PAGE (6 - 12% BOLT, ThermoFisher), and the protein-containing fractions were pooled and concentrated.

[0177] The protein was further purified by size exclusion chromatography (SEC) (Superdex 200, ThermoFisher) using SEC buffer (40 mM Tris, 7.5; 20 mM MgCl 2 , 150 mM NaCl), and the fractions were analyzed by SDS-PAGE (6 - 12% BOLT, ThermoFisher). Protein-containing fractions were pooled according to their appearance and analyzed for activity using the ITK kinase enzyme system combined with the ADP-Glo™ assay (Promega) according to the manufacturer's instructions. Briefly, full-length ITK expressed from EG17 and EG18 was used in the assay at total enzyme concentrations of 200 ng, 100 ng, 50 ng, and 0 ng. Substrate PolyE4Y1 was used at a concentration of 0.2 μg / μl, and ATP was added to the reaction at 25 μM. In a 96-well plate, 5 μl of reaction buffer (supplied with the kit) was combined with 10 μl of enzyme dilution and 10 μl of ATP / PolyE4Y1 mix. The plate was incubated at RT for 60 minutes. 25 μl The ADP-Glo reagent was added and the plate was incubated again at RT for 40 minutes. The reaction was stopped by adding 50 μl of kinase detection reagent and incubating for an additional 30 minutes at RT. The reaction was read by luminescence with an integration time of 1 second.

[0178] Figure 16 shows the purification process for the ITK protein and the ITK protein fused to the enhancer protein L. In purification using SEC, two peaks (P1 and P2) can be identified as the target protein that can be identified by Western blot as monomer (P2) and dimer (P1) species (data not shown). Without being bound by theory, ITK appears to need to form a dimer to achieve the active form. ITK is a known kinase that is toxic to cells when overexpressed. Thus, as the activity of ITK increases, more expression will be downregulated by the host cell or made into the monomeric inactive form.

[0179] Figure 17A shows the final SDS-PAGE of the purification of the identified species. Note that only the P1 species is active, and thus the expression of the enhancer protein in combination with ITK results in a significant increase in the expression of the active ITK species. Figure 17B demonstrates the difference in activity by using luminescence as the primary readout. Only the P1 expressed from EG17 demonstrates high activity and is thus the only protein available for use in drug screening for this kinase. Although both systems appear to express similar amounts of the protein of interest, the ITK expressed using the methods disclosed herein exhibits greater activity than the ITK protein expressed in the absence of the enhancer protein. This example demonstrates that using the methods disclosed herein, it is possible to produce active proteins that would otherwise be toxic or inactivated by the host cell without using this method. Furthermore, using the disclosed methods, not only can active proteins that would otherwise be toxic be produced, but these proteins can then be used in drug screening such as small molecule screening to discover novel therapeutics.

[0180] (Example 9) Production of Interleukin-2 Inducible T-Cell Kinase (ITK) in CHO-K1 Cells The experiment of Example 8 was repeated using CHO cells instead of HEK293. ITK-his was expressed from EG17 or the control construct EG18.

[0181] An ITK-his fusion was expressed in CHO-K1 cells. Prior to transiently transfecting either EG17 or EG18 using Lipofectamine 3000 (Thermofisher) according to the manufacturer's instructions, a total of eight 150 mm plates of CHO-K1 cells for each construct were seeded at 5×10 6 cells per dish and incubated at 37 °C and 5% CO 2Incubated overnight at [temperature]. Cells were collected 48 hours later by scraping, spun down, and the supernatant removed (5,000×g, 15 minutes, 4°C). The cell pellet was stored at -80°C until further use. To purify ITK, cells were resuspended in lysis buffer (40 mM Tris, 7.5; 20 mM MgCl 2 ; 0.1 mg / ml BSA; 50 μM DTT; and 2 mM MnCl 2 , protease inhibitor, DNAse), lysed by sonication (2 minutes, 10 seconds on, 10 seconds off, 40% amplitude), and the crude cell extract clarified (5,000×g, 20 minutes, 4°C). Before loading the clarified lysate using a peristaltic pump, a 5 ml His-resin column (GE Healthcare HisTrap) was equilibrated with wash buffer (40 mM Tris, 7.5; 20 mM MgCl2; 0.1 mg / ml BSA; 50 μM DTT; and 2 mM MnCl2). After loading, purification was performed in an AKTA system. The column was washed with 5 CV of wash buffer before eluting over 20 CV with a continuous gradient of 0 - 75% elution buffer (wash buffer + 300 mM imidazole). Elution was completed with 5 CV of 100% elution buffer.

[0182] Protein-containing fractions were analyzed by SDS-PAGE (6 - 12% SurePAGE, Bis-Tris, GenScript), the protein-containing fractions pooled and concentrated. The protein was further purified by size exclusion chromatography (SEC) (Superdex 200, ThermoFisher) using SEC buffer (40 mM Tris, 7.5; 20 mM MgCl 2 , 150 mM NaCl), and the fractions analyzed by SDS-PAGE (6 - 12% SurePAGE, Bis-Tris, GenScript). Protein-containing fractions were pooled according to their appearance and analyzed for activity using an ITK kinase enzyme system combined with the ADP-Glo assay (trademark) (Promega) according to the manufacturer's instructions.

[0183] ΔITK expressed in Sf9 insect cells was used as a standard. Full-length ITK was used in the assay together with ΔITK expressed from EG17 and EG18 at total enzyme concentrations of 200 ng, 100 ng, 50 ng, and 0 ng. Substrate PolyE4Y1 was used at a concentration of 0.2 μg / μl, and ATP was added to the reactants at 25 μM. In a 96-well plate, 5 μl of reaction buffer (supplied with the kit) was combined with 10 μl of enzyme dilution and 10 μl of ATP / PolyE4Y1 mix. The plate was incubated at RT for 60 minutes. 25 μl of ADP-Glo reagent was added, and the plate was incubated again at RT for 40 minutes. The reaction was stopped by adding 50 μl of kinase detection reagent and incubating for an additional 30 minutes at RT. The reaction was read by luminescence with an integration time of 1 second.

[0184] Figure 18 shows the purification process of ITK expressed with and without enhancer protein L. As mentioned above, in the purification using SEC, two peaks (P1 and P2) can be identified as the target proteins. Without being bound by theory, ITK seems to need to form dimers to achieve the active form. ITK is a known kinase that is toxic to cells when overexpressed. Thus, as the activity of ITK increases, more expression will be downregulated by the host cell or made into the monomeric inactive form.

[0185] Figure 19 demonstrates the difference in activity by using luminescence as the primary readout. Only P1 expressed from EG17 demonstrates comparable activity to the provided ΔITK positive control. Although both systems seem to express similar amounts of the protein of interest, only the presented system achieves the production of active protein by controlling host cell regulation. This example demonstrates that the methods disclosed herein can be used to produce active proteins that would otherwise be toxic or inactivated by the host cell without using these methods.

[0186] (Example 10) Production of interleukin-2-inducible T-cell kinase (ITK) in Sf9 cells Repeat Example 8 using Sf9 cells instead of HEK293 cells. Express ITK-his from the EG17 construct or the industrial and academic standard EG18 construct. Expression in Sf9 cells is performed as described in Example 7, and protein purification of the His-tagged ITK protein is performed as described in Examples 8 and 9.

[0187] (Example 11) Expression of cystic fibrosis transmembrane conductance regulator (CFTR) CFTR was used as an additional example to demonstrate that co-expression of a membrane protein as a target protein in combination with a pore-blocking protein as an enhancer protein resulted in a high density of active ion channels. CFTR is a transmembrane transporter of the ABC transporter class that conducts chloride ions across the epithelial cell membrane. CFTR is known to be expressed in a heterogeneous form when using the academic standard (EG24). Heterogeneity increases the difficulty in the purification or analysis of ABC transporters. To demonstrate an improvement in homogeneity, CFTR was cloned into the backbone of the exemplary system (EG25) or used as a PCR product. As a comparison, the academic standard (EG24) was used in parallel as a control.

[0188] Express the CFTR construct in HEK293 cells. Before transiently transfecting any of EG25, the PCR product of the EG25 insert, or EG24 as described above, seed HEK293 cells at 0.3 × 10 6 cells / well in a 6-well plate and incubate at 37 °C and 5% CO 2It was incubated overnight. CFTR expression was monitored using a microscope 24 and 48 hours later. Cells were collected 48 hours later and lysed using RIPA (radioimmunoprecipitation assay) buffer (CellGene). The lysate was clarified and analyzed by SDS-PAGE (6 - 12% BOLT, ThermoFisher) followed by Western blot (nitrocellulose membrane, ThermoFisher) using anti-CFTR (Abcam, secondary antibody - anti-mouse-HRP).

[0189] Figure 7 demonstrates the effect of co-expression of L-protein and CFTR. While the academic standard produced a broad band in the Western blot, transcription and translation based on the EG25 construct resulted in a defined band, demonstrating a highly homogeneous expression of the ABC transporter. Moreover, this example demonstrates that the expression system can be delivered into cells as a vector or PCR product.

[0190] (Example 12) Expression of NADase Using NADase as an exemplary target protein, the application of the disclosed system to toxic soluble proteins that are difficult to express was exemplified. NADase is an enzyme protein that catalyzes the reaction of NAD+ to ADP-ribose and nicotinamide. Overexpression of NADase usually results in increased cell death due to the fact that cells are deprived of their natural energy source NAD+. To demonstrate that this system can produce a high yield of active NADase, an NADase-Flag fusion was cloned into the backbone of the exemplary system (EG13).

[0191] The NADase-flag construct was expressed in HEK293 cells. Prior to transiently transfecting any of EG13 as described above, HEK293 cells were seeded at 5×10 6 cells in a T225 flask and incubated at 37°C and 5% CO 2Incubated overnight. NADase-flag expression was monitored using a microscope at 24 and 48 hours. Cells were detached using 0.5% trypsin solution at 37 °C for 5 minutes and collected at 48 hours by scraping. Cells were pelleted (5,000×g, 15 minutes, 4 °C) and the supernatant was discarded. Cell pellets were stored at -80 °C until further use. To purify NADase-flag, cells were resuspended in lysis buffer (50 mM NaHPO4 pH 8.0, 300 mM NaCl, 0.01% Tween20, protease inhibitor, DNAse), lysed by sonication (2 minutes, 10 seconds on, 10 seconds off, 40% amplitude), and the crude cell extract was clarified (100,000×g, 45 minutes, 4 °C). Anti-FLAG M2 affinity gel (Sigma) was equilibrated with wash buffer (50 mM NaHPO4 pH 8.0, 300 mM NaCl, 0.01% Tween20) before adding to the clarified lysate. The lysate was incubated with the resin for 2 hours at 4 °C with shaking. The resin was pelleted and washed with 5 CV of wash buffer, and the protein was eluted using a spin column with 4 × 1 CV of elution buffer (wash buffer + 0.2 mg / ml 3×Flag-peptide (Sigma)). Purification was analyzed by SDS-PAGE (6 - 12% BOLT, ThermoFisher) (Figure 8A), and protein-containing fractions were pooled. Protein concentration was measured using A280 (NanoDrop One, Fisher Scientific). The protein yield was determined to be 26 mg / L of expression medium. The activity of NADase was examined by analyzing the conversion rate from NAD+ to ADP-ribose by HPLC (Figure 8B).

[0192] (Example 13) Production of the secreted protein, C1-esterase inhibitor (C1-Inh) Using C1-Inh as an exemplary target protein, the application of the disclosed method for expressing a secreted protein with accurate post-translational modifications was illustrated. C1-Inh is a protease inhibitor belonging to the serpin superfamily. As a secreted protein, C1-Inh is highly glycosylated and thus proves to be a difficult target for recombinant expression. The C1-Inh-myc-flag fusion protein was expressed in the presence or absence of the EMCV-derived L protein expressed from separate constructs. In this example, the EMCV-derived L protein was co-expressed under the control of the CMV promoter from separate constructs.

[0193] The C1-Inh-Myc-Flag fusion was expressed in HEK293 cells. Prior to transient transfection of the vector encoding C1-Inh (OriGene; CAT#: RC203767) alone or in combination with EG11 by transfection of suspension cells using methods known in the art and / or disclosed herein, HEK293 cells were seeded at 1.75×10 6 cells / ml in a 100 ml shake flask and incubated at 37 °C, 5% CO 2And incubated overnight at 120 rpm. The supernatant containing the expressed recombinant C1-Inh protein was collected after 72 hours, and the supernatant was clarified by centrifugation followed by filtration (22 µm, nitrocellulose). Before adding to the supernatant, to purify C1-Inh, anti-Flag resin (anti-FLAG M2 affinity gel, Millipore Sigma) was equilibrated with 20 mM Tris pH 7.5, 50 mM NaCl. The supernatant was incubated with the resin for 2 hours at 4 °C while shaking. The resin was sedimented, washed with 5 column volumes (CV) of 20 mM Tris pH 7.5, 50 mM NaCl, and the protein was eluted with 4 CV of 20 mM Tris pH 7.5, 50 mM NaCl, 0.2 mg / ml 3×Flag peptide. The purification was analyzed by SDS-PAGE (SurePAGE, Bis-Tris, GenScript), and the protein-containing fractions were pooled. The protein concentration was analyzed by BCA assay (ThermoFisher) according to the manufacturer's instructions, and the normalized C1-Inh was assayed for activity using an immunoassay (MicroVue C1-Inihibitor Plus EIA, Quidel) according to the manufacturer's instructions.

[0194] Figure 20A shows the purification of C1-inhibitor in the absence (left) and presence (right) of enhancer protein. The total amount of C1-inhibitor produced is increased by >30% in the presence of enhancer protein. Figure 20B demonstrates the improvement in the total amount of active C1-inhibitor in the purified sample. Before assaying for active C1-inhibitor, the protein concentration was normalized for the activity assay. The amount of active C1-inhibitor can be increased by >10% by co-expressing the enhancer protein simultaneously with the gene of interest (GOI). These results demonstrate that the methods disclosed herein result in higher yields and improved activity of secreted target proteins such as C1-inhibitor.

[0195] (Example 14) Production of the secreted protein, pregnancy-specific glycoprotein 1 (PSG1) Using PSG1 as an exemplary target protein, the application of the disclosed method for expressing a secreted protein with accurate post-translational modifications was illustrated. PSG1 is a highly glycosylated secreted protein of the human PSG family within the carcinoembryonic antigen superfamily. PSG1 is one of the most abundant fetal proteins found in maternal blood during pregnancy. PSG1 has been shown to function as an immunomodulatory agent by upregulating TGF-beta in macrophages, monocytes, and trophoblasts. In addition, PSG1 has been shown to induce the secretion of the anti-inflammatory cytokines IL-10 and IL-6 in human monocytes. These functions have made PSG1 an attractive pharmaceutical target. The difficulty in expressing PSG1 is the correct glycosylation pattern that cannot be reconstituted when using non-human cells. In this example, the L protein derived from EMCV was co-expressed with PSG1 under the control of the CMV promoter.

[0196] PSG1 was expressed in HEK293 cells. Prior to transiently transfecting a vector encoding PSG1 in cooperation with the L protein derived from EMCV, HEK293 cells were seeded at 1.75×10 6 cells / ml in a 100 ml shaking flask and incubated at 37 °C, 5% CO 2And incubated overnight at 120 rpm. The supernatant containing the expressed recombinant PSG1 protein was collected after 72 hours, and the supernatant was clarified by centrifugation followed by filtration (22 um, nitrocellulose). Before loading the supernatant onto the column using a peristaltic pump, the HiTrap™ DEAE Sepharose Fast Flow IEX column (Cytiva (formerly GE Healthcare Life Sciences)) was equilibrated with a washing buffer (10 mM Tris pH 7.6) to purify PSG1. After loading, purification was performed on an AKTA™ system (Cytiva Life Sciences (formerly GE Healthcare)). Before eluting with a multi-step gradient of 10%, 20%, 30%, 50% and 100% elution buffer (washing buffer + 200 mM NaCl), the column was washed with 5 CV of washing buffer. Protein-containing fractions were pooled, concentrated, and analyzed by SDS-PAGE (6 - 12% BOLT, ThermoFisher) and Western blot (nitrocellulose membrane, ThermoFisher) using anti-PSG1 (Invitrogen, secondary antibody - anti-rabbit-HRP).

[0197] Figure 21 shows the ion exchange chromatography of PSG1 (left). Protein-containing fractions (Figure 21A, red box) were pooled and concentrated before confirming the presence and identity of PSG1 by SDS-PAGE and Western blot (Figure 21B, red arrow).

[0198] Further numbered embodiments Further embodiments of the present invention are provided in the numbered embodiments below:

[0199] Embodiment 1. A system for the recombinant expression of a target protein in a eukaryotic cell, comprising one or more vectors, wherein the one or more vectors comprise a. a first polynucleotide encoding the target protein, and b. a second polynucleotide encoding an enhancer protein and i. the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or ii. the enhancer protein is selected from the group consisting of picornavirus leader (L) protein, picornavirus 2A protease, rhinovirus 3C protease, herpes simplex virus (HSV) ICP27 protein, and rhabdovirus matrix (M) protein, A system wherein the first polynucleotide and the second polynucleotide are operably linked to one or more promoters.

[0200] Embodiment 2. The system according to Embodiment 1, wherein the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT).

[0201] Embodiment 3. The system according to Embodiment 2, wherein the NCT inhibitor is a viral protein.

[0202] Embodiment 4. The system according to any one of Embodiments 1 to 3, wherein the NCT inhibitor is selected from the group consisting of picornavirus leader (L) protein, picornavirus 2A protease, rhinovirus 3C protease, coronavirus ORF6 protein, Ebola virus VP24 protein, Venezuelan equine encephalitis virus (VEEV) capsid protein, herpes simplex virus (HSV) ICP27 protein, and rhabdovirus matrix (M) protein.

[0203] Embodiment 5. The system according to Embodiment 4, wherein the NCT inhibitor is picornavirus leader (L) protein or a functional variant thereof.

[0204] Embodiment 6. The system according to Embodiment 4, wherein the NCT inhibitor is picornavirus 2A protease or a functional variant thereof.

[0205] Embodiment 7. The system according to Embodiment 4, wherein the NCT inhibitor is a rhinovirus 3C protease or a functional variant thereof.

[0206] Embodiment 8. The system according to Embodiment 4, wherein the NCT inhibitor is a coronavirus ORF6 protein or a functional variant thereof.

[0207] Embodiment 9. The system according to Embodiment 4, wherein the NCT inhibitor is an Ebola virus VP24 protein or a functional variant thereof.

[0208] Embodiment 10. The system according to Embodiment 4, wherein the NCT inhibitor is a Venezuelan equine encephalitis virus (VEEV) capsid protein or a functional variant thereof.

[0209] Embodiment 11. The system according to Embodiment 4, wherein the NCT inhibitor is a herpes simplex virus (HSV) ICP27 protein or a functional variant thereof.

[0210] Embodiment 12. The system according to Embodiment 4, wherein the NCT inhibitor is a rhabdovirus matrix (M) protein or a functional variant thereof.

[0211] Embodiment 13. The system according to Embodiment 5, wherein the L protein is the L protein of Theiler's virus or a functional variant thereof.

[0212] Embodiment 14. The system according to Embodiment 5, wherein the L protein shares at least 90% identity with SEQ ID NO: 1.

[0213] Embodiment 15. The system according to Embodiment 5, wherein the L protein is the L protein of encephalomyocarditis virus (EMCV) or a functional variant thereof.

[0214] Embodiment 16. The system according to embodiment 5, wherein the L protein shares at least 90% identity with SEQ ID NO: 2.

[0215] Embodiment 17. The system according to embodiment 5, wherein the L protein is selected from the group consisting of the L protein of poliovirus, the L protein of HRV16, the L protein of mengovirus, and the L protein of Sapporo virus 2, or a functional variant thereof.

[0216] Embodiment 18. The system according to any one of embodiments 1 to 17, wherein the system comprises a single vector containing an expression cassette, and the expression cassette contains the first polynucleotide and the second polynucleotide.

[0217] Embodiment 19. The system according to embodiment 18, wherein the expression cassette contains a first promoter operably linked to the first polynucleotide and a second promoter operably linked to the second polynucleotide.

[0218] Embodiment 20. The system according to embodiment 18, wherein the expression cassette contains a shared promoter operably linked to both the first polynucleotide and the second polynucleotide.

[0219] Embodiment 21. The system according to embodiment 20, wherein the expression cassette contains a coding polynucleotide containing the first polynucleotide and the second polynucleotide linked by a polynucleotide encoding a ribosome skipping site, and the coding polynucleotide is operably linked to the shared promoter.

[0220] Embodiment 22. The system according to embodiment 20, wherein the expression cassette contains a coding polynucleotide, the coding polynucleotide encodes the enhancer protein and the target protein linked by a ribosome skipping site, and the coding polynucleotide is operably linked to the shared promoter.

[0221] Embodiment 23. The system according to any one of embodiments 18 to 22, wherein the expression cassette is configured for the transcription of a single messenger RNA encoding both the target protein and the enhancer protein linked by a ribosome skipping site, and the translation of the messenger RNA results in the expression of the target protein and the L protein as separate polypeptides.

[0222] Embodiment 24. The system according to any one of embodiments 1 to 23, comprising one type of vector.

[0223] Embodiment 25. a. A first vector comprising the first polynucleotide operably linked to a first promoter; b. A second vector comprising the second polynucleotide operably linked to a second promoter The system according to any one of embodiments 1 to 17.

[0224] Embodiment 26. The system according to any one of embodiments 1 to 17 or embodiment 25, comprising two types of vectors.

[0225] Embodiment 27. The system according to any one of embodiments 1 to 26, wherein either or both of the first polynucleotide and the second polynucleotide are operably linked to an internal ribosome entry site (IRES).

[0226] Embodiment 28. The system according to any one of Embodiments 1 to 27, wherein at least one of the one or more vectors contains a T7 promoter configured for transcription of either or both of the first polynucleotide and the second polynucleotide by T7 RNA polymerase.

[0227] Embodiment 29. The system according to any one of Embodiments 1 to 28, wherein at least one of the one or more vectors contains a polynucleotide sequence encoding T7 RNA polymerase.

[0228] Embodiment 30. a. A first polynucleotide encoding a target protein, b. A second polynucleotide encoding an enhancer protein A vector for recombinant expression of a target protein in a eukaryotic cell, comprising: i. the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or ii. the enhancer protein is selected from the group consisting of picornavirus leader (L) protein, picornavirus 2A protease, rhinovirus 3C protease, coronavirus ORF6 protein, Ebola virus VP24 protein, Venezuelan equine encephalitis virus (VEEV) capsid protein, herpes simplex virus (HSV) ICP27 protein, and rhabdovirus matrix (M) protein, The first polynucleotide and the second polynucleotide are operably linked to at least one promoter.

[0229] Embodiment 31. The vector according to Embodiment 30, wherein the expression cassette contains a first promoter operably linked to the first polynucleotide and a second promoter operably linked to the second polynucleotide.

[0230] Embodiment 32. The vector according to Embodiment 30, wherein the expression cassette comprises a shared promoter operably linked to both the first polynucleotide and the second polynucleotide.

[0231] Embodiment 33. A eukaryotic cell for the expression of a target protein, comprising an exogenous polynucleotide encoding an enhancer protein, a. the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or b. the enhancer protein is selected from the group consisting of picornavirus leader (L) protein, picornavirus 2A protease, rhinovirus 3C protease, coronavirus ORF6 protein, Ebola virus VP24 protein, Venezuelan equine encephalitis virus (VEEV) capsid protein, herpes simplex virus (HSV) ICP27 protein, and rhabdovirus matrix (M) protein, wherein the exogenous polynucleotide is operably linked to a promoter.

[0232] Embodiment 34. The eukaryotic cell according to Embodiment 33, wherein the polynucleotide is operably linked to an internal ribosome entry site (IRES).

[0233] Embodiment 35. The eukaryotic cell according to Embodiment 33 or Embodiment 34, wherein the promoter is an inducible promoter.

[0234] Embodiment 36. A method for the recombinant expression of a target protein, comprising introducing a polynucleotide encoding the target protein, operably linked to a promoter, into the cell according to any one of Embodiments 33 to 35.

[0235] Embodiment 37. A method for recombinant expression of a target protein, comprising the step of introducing the system according to any one of Embodiments 1 to 29 or the vector according to any one of Embodiments 30 to 32 into a eukaryotic cell.

[0236] Embodiment 38. The method according to Embodiment 36 or 37, wherein the target protein is a membrane protein.

[0237] Embodiment 39. The method according to Embodiment 38, wherein the localization of the membrane protein to the cell membrane is increased as compared to the localization observed when the membrane protein is expressed without the enhancer protein.

[0238] Embodiment 40. A eukaryotic cell produced by introduction of the system according to any one of Embodiments 1 to 29 or the vector according to any one of Embodiments 30 to 32.

[0239] Embodiment 41. A target protein expressed by introduction of the system according to any one of Embodiments 1 to 29 or the vector according to any one of Embodiments 30 to 32 into a eukaryotic cell.

[0240] Embodiment 42. A method for expressing a target protein in a eukaryotic cell, comprising the step of introducing into the eukaryotic cell a polynucleotide encoding the target protein operably linked to a promoter, wherein the method utilizes co-expression of an enhancer protein to enhance the expression level, solubility and / or activity of the target protein, (a) the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or (b) The method wherein the enhancer protein is selected from the group consisting of picornavirus leader (L) protein, picornavirus 2A protease, rhinovirus 3C protease, coronavirus ORF6 protein, Ebola virus VP24 protein, Venezuelan equine encephalitis virus (VEEV) capsid protein, herpes simplex virus (HSV) ICP27 protein, and rhabdovirus matrix (M) protein.

[0241] Embodiment 43. The method according to embodiment 42, wherein the co-expression of the enhancer protein comprises introducing into the eukaryotic cell a polynucleotide encoding the enhancer protein operably linked to a promoter.

[0242] Embodiment 44. The method according to embodiment 42 or embodiment 43, wherein the introducing step(s) comprises transfection of the eukaryotic cell with one or more DNA molecules, transduction of the eukaryotic cell with a single viral vector, and / or transduction of the eukaryotic cell with two viral vectors.

[0243] Embodiment 45. The system according to any one of embodiments 1 to 29, the vector according to any one of embodiments 30 to 32, the eukaryotic cell according to any one of embodiments 33 to 35, the method according to any one of embodiments 36 to 39 and 42 to 44, the eukaryotic cell according to embodiment 40, and the target protein according to embodiment 41, wherein the target protein is a soluble protein.

[0244] Embodiment 46. The system according to any one of embodiments 1 to 29, the vector according to any one of embodiments 30 to 32, the cell according to any one of embodiments 33 to 35, or the method according to any one of embodiments 36 to 44, wherein the target protein is a secreted protein.

[0245] Embodiment 47. The system according to any one of Embodiments 1 to 29, the vector according to any one of Embodiments 30 to 32, the eukaryotic cell according to any one of Embodiments 33 to 35, the method according to any one of Embodiments 36 to 39 and 42 to 44, the eukaryotic cell according to Embodiment 40, and the target protein according to Embodiment 41, wherein the target protein is a membrane protein.

[0246] Embodiment 48. The system according to any one of Embodiments 1 to 29, the vector according to any one of Embodiments 30 to 32, the eukaryotic cell according to any one of Embodiments 33 to 35, the method according to any one of Embodiments 36 to 39 and 42 to 44, the eukaryotic cell according to Embodiment 40, and the target protein according to Embodiment 41, wherein the target protein is dopamine receptor D1 (DRD1), and optionally, the DRD1 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 19.

[0247] Embodiment 49. The system according to any one of Embodiments 1 to 29, the vector according to any one of Embodiments 30 to 32, the eukaryotic cell according to any one of Embodiments 33 to 35, the method according to any one of Embodiments 36 to 39 and 42 to 44, the eukaryotic cell according to Embodiment 40, and the target protein according to Embodiment 41, wherein the target protein is cystic fibrosis transmembrane conductance regulator (CFTR), and optionally, the CFTR comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 18.

[0248] Embodiment 50. The target protein is C1 esterase inhibitor (C1-Inh), and optionally, the C1-Inh comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 16. The system according to any one of Embodiments 1 to 29, the vector according to any one of Embodiments 30 to 32, the eukaryotic cell according to any one of Embodiments 33 to 35, the method according to any one of Embodiments 36 to 39 and 42 to 44, the eukaryotic cell according to Embodiment 40, and the target protein according to Embodiment 41.

[0249] Embodiment 51. The target protein is ITK, and optionally, the ITK comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 15. The system according to any one of Embodiments 1 to 29, the vector according to any one of Embodiments 30 to 32, the eukaryotic cell according to any one of Embodiments 33 to 35, the method according to any one of Embodiments 36 to 39 and 42 to 44, the eukaryotic cell according to Embodiment 40, and the target protein according to Embodiment 41.

[0250] Embodiment 52. The target protein is NADase, and optionally, the NADase comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 20. The system according to any one of Embodiments 1 to 29, the vector according to any one of Embodiments 30 to 32, the eukaryotic cell according to any one of Embodiments 33 to 35, the method according to any one of Embodiments 36 to 39 and 42 to 44, the eukaryotic cell according to Embodiment 40, and the target protein according to Embodiment 41.

[0251] Embodiment 53. A method for generating an antibody against a target protein, the method comprising immunizing a subject with the cell according to any one of Embodiments 33 to 35, the cell according to Embodiment 40, or the target protein according to Embodiment 41.

[0252] Embodiment 54. The method according to Embodiment 53, further comprising the step of isolating one or more immune cells that express an immunoglobulin protein specific for the target protein.

[0253] Embodiment 55. The method according to Embodiment 53 or Embodiment 54, comprising the step of generating one or more hybridomas from the one or more immune cells.

[0254] Embodiment 56. The method according to any one of Embodiments 53 to 55, comprising the step of cloning one or more immunoglobulin genes from the one or more immune cells.

[0255] Embodiment 57. A method for antibody discovery by cell sorting, comprising: a. The cell according to any one of Embodiments 33 to 35, the eukaryotic cell according to Embodiment 40, or the target protein according to Embodiment 41, wherein the cell or the target protein is labeled, and b. A step of preparing a solution comprising a population of recombinant cells that express a library of polypeptides each containing an antibody or an antigen-binding fragment thereof, and isolating one or more recombinant cells from the solution by sorting the labeled cells or the recombinant cells bound to the labeled target protein.

[0256] Embodiment 58. A method for panning a phage display library, comprising: a. A step of mixing a phage display library with the eukaryotic cell according to any one of Embodiments 33 to 35, the eukaryotic cell according to Embodiment 40, or the target protein according to Embodiment 41, and b. A step of purifying and / or enriching members of the phage display library that bind to the cell or the target protein.

[0257] Embodiment 59. The eukaryotic cell according to any one of Embodiments 33 to 35 and 40, which is a human cell, an animal cell, an insect cell, a plant cell, or a fungal cell.

[0258] Embodiment 60. The eukaryotic cell according to any one of Embodiments 33 to 35, 40, and 59, which is a eukaryotic cell line.

[0259] Embodiment 61. The eukaryotic cell according to any one of Embodiments 33 to 35, 40, 59, and 60, which is Bc HROC277, COS, CHO, CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV, VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO, 5P2 / 0-Ag14, HeLa, HEK293, HEK293-F, HEK293-H, HEK293-T, perC6 cell, Sf9 cell, Saccharomyces cell, Pichia cell, or Schizosaccharomyces cell.

[0260] Embodiment 62. The eukaryotic cell according to Embodiment 60, wherein the eukaryotic cell line is a stable cell line.

[0261] Embodiment 63. The system according to any one of Embodiments 1 to 29 and 45 to 52, wherein the one or more vectors are selected from the group consisting of an adeno-associated virus (AAV) vector, a lentivirus vector, a retrovirus vector, a replication-competent adenovirus vector, a replication-deficient adenovirus vector, a herpesvirus vector, a baculovirus vector, or a non-viral plasmid.

[0262] Embodiment 64. The system according to Embodiment 63, wherein at least one of the one or more vectors is an AAV vector.

[0263] Embodiment 65. The vector according to any one of Embodiments 30 to 32, which is an adeno-associated virus (AAV) vector, a lentiviral vector, a retroviral vector, a replication-competent adenoviral vector, a replication-deficient adenoviral vector, a herpesvirus vector, a baculovirus vector, or a non-viral plasmid.

[0264] Embodiment 66. The vector according to Embodiment 65, which is an AAV vector.

[0265] Embodiment 67. The system according to Embodiment 4, wherein the rhabdovirus matrix (M) protein is the M protein of vesicular stomatitis virus (VSV).

[0266] Embodiment 68. The system according to Embodiment 67, wherein the M protein shares at least 90% identity with SEQ ID NO: 9.

[0267] Embodiment 69. A system for recombinant expression of a target protein in a eukaryotic cell, comprising one or more vectors, wherein the one or more vectors a. a first polynucleotide encoding the target protein, and b. a second polynucleotide encoding the L protein of encephalomyocarditis virus (EMCV), wherein, optionally, the L protein shares at least 90% identity with SEQ ID NO: 2, the second polynucleotide and the first polynucleotide and the second polynucleotide are operably linked to one or more promoters.

[0268] Embodiment 70. A system for recombinant expression of a target protein in a eukaryotic cell, comprising one or more vectors, wherein the one or more vectors a. a first polynucleotide encoding the target protein, and b. A second polynucleotide encoding the L protein of the Tyler virus, wherein, optionally, the L protein shares at least 90% identity with SEQ ID NO: 1, and the second polynucleotide and the first polynucleotide and the second polynucleotide are operably linked to one or more promoters. A system

[0269] Embodiment 71. A system for recombinant expression of a target protein in a eukaryotic cell, comprising one or more vectors, wherein the one or more vectors a. A first polynucleotide encoding the target protein, and b. A second polynucleotide encoding a picornavirus 2A protease, wherein, optionally, the picornavirus 2A protease shares at least 90% identity with SEQ ID NO: 7, and the second polynucleotide and the first polynucleotide and the second polynucleotide are operably linked to one or more promoters. A system

[0270] Embodiment 72. A system for recombinant expression of a target protein in a eukaryotic cell, comprising one or more vectors, wherein the one or more vectors a. A first polynucleotide encoding the target protein, and b. A second polynucleotide encoding the M protein of vesicular stomatitis virus (VSV), wherein, optionally, the M protein shares at least 90% identity with SEQ ID NO: 9, and the second polynucleotide and the first polynucleotide and the second polynucleotide are operably linked to one or more promoters. A system

[0271] Embodiment 73. The target protein is dopamine receptor 1 (DRD1), and optionally, the DRD1 includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 19. The system according to any one of Embodiments 69 to 72.

[0272] Embodiment 74. The target protein is cystic fibrosis transmembrane conductance regulator (CFTR), and optionally, the CFTR includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 18. The system according to any one of Embodiments 69 to 72.

[0273] Embodiment 75. The target protein is C1 esterase inhibitor (C1-Inh), and optionally, the C1-Inh includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 16. The system according to any one of Embodiments 69 to 72.

[0274] Embodiment 76. The target protein is ITK, and optionally, the ITK includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 15. The system according to any one of Embodiments 69 to 72.

[0275] Embodiment 77. The target protein is NADase, and optionally, the NADase includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 20. The system according to any one of Embodiments 69 to 72.

Claims

[Claim 1] The invention described in the specification.

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