Systems and methods for protein expression

Abstract

The present disclosure provides a system for expressing a target protein in conjunction with an enhancer protein. The enhancer protein can be a viral protein that blocks nucleocytoplasmic transport. Polynucleotides, vectors, and cells comprising nucleic acid sequences of the target protein and the enhancer protein are also provided. In one aspect, the present disclosure provides a system for recombinantly expressing a target protein in a eukaryotic cell, comprising one or more vectors. The vector(s) comprises a first polynucleotide encoding the target protein and a second polynucleotide encoding the enhancer protein.

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 September 16, 2019, and U.S. Provisional Patent Application No. 62 / 970,628, filed February 5, 2020, the entire contents of each of which are incorporated by reference herein for all purposes.

[0002] Incorporating sequence tables The contents of the text file submitted electronically attached hereto are incorporated by reference in their entirety: Computer-readable format copy of the sequence listing (Filename: EXCI_001_02WO_SeqList_ST25.txt, Recorded on: September 15, 2020, File Size: 84.8kb). [Background technology]

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

[0004] There remains an unmet need for compositions and methods useful in the production of recombinant proteins. Summary of the Invention [Means for solving the problem]

[0005] overview The inventors have recognized that co-expression of a target protein with a certain enhancer protein improves recombinantly produced protein. 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 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 target proteins that exhibit improved properties (e.g., reduced misfolding, altered activity, incorrect post-translational modification and / or toxicity); (4) the disclosed compositions and methods increase the correct folding and / or high yield of recombinant proteins; (5) the disclosed compositions and methods improve the performance of downstream activation pathways (e.g., GPCR signaling); and / or (6) the co-expression of the enhancer protein does not affect the functionality of the target protein and / or the downstream metabolism of the cell. The invention is not limited by these listed advantages, as some embodiments may exhibit none, some, or all of these advantages.

[0006] In one aspect, the present disclosure provides a system for recombinant expression of target protein in eukaryotic cells, comprising one or more vectors.The vector(s) comprises 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 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 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 recombinant expression of a target protein, comprising introducing a vector system of the present disclosure into a eukaryotic cell. In yet another aspect, the present disclosure provides a cell produced by introducing a 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 a 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, comprising introducing a polynucleotide (operably linked to a promoter) encoding the target protein into the eukaryotic cell. 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 antibodies against a target protein, comprising immunizing a subject with a cell or target protein produced using a system or method of the present disclosure. In yet another aspect, the present disclosure provides a method for antibody discovery by cell sorting, comprising providing a solution comprising a population of labeled cells or labeled target proteins produced using a system or method of the present disclosure and recombinant cells, wherein the recombinant cells express a library of polypeptides comprising antibodies or antigen-binding fragments thereof, respectively, and sorting one or more recombinant cells from the solution by detecting the labeled cells or recombinant cells bound to the labeled target proteins. In a further aspect, the present disclosure provides a method for panning a phage display library, comprising mixing the phage display library with cells or target proteins produced using a system or method of the present disclosure, and purifying and / or enriching members of the phage display library that bind to the cells or target proteins.

[0009] Further aspects and embodiments are provided in the detailed disclosure that follows, and the invention is not limited by this Summary. In an embodiment 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, said one or more vectors comprising: a) a first polynucleotide encoding the target protein; b) a second polynucleotide encoding an enhancer protein; Including, 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; The system, wherein the first polynucleotide and the second polynucleotide are operably linked to one or more promoters. (Item 2) The system described in item 1, wherein the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT). (Item 3) 3. The system of claim 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 a picornavirus leader (L) protein or a functional variant thereof. (Item 6) The system of item 4, wherein the NCT inhibitor is picornavirus 2A protease or a functional variant thereof. (Item 7) The system of item 4, wherein the NCT inhibitor is rhinovirus 3C protease or a functional variant thereof. (Item 8) The system of item 4, wherein the NCT inhibitor is a coronavirus ORF6 protein or a functional variant thereof. (Item 9) 5. The system of claim 4, wherein the NCT inhibitor is an Ebola virus VP24 protein or a functional variant thereof. (Item 10) 5. The system of claim 4, wherein the NCT inhibitor is a Venezuelan Equine Encephalitis Virus (VEEV) capsid protein or a functional variant thereof. (Item 11) The system of item 4, wherein the NCT inhibitor is 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) 6. The system of item 5, wherein the L protein is the L protein of Theiler's virus or a functional variant thereof. (Item 14) The system described in item 5, wherein the L protein shares at least 90% identity with SEQ ID NO:1. (Item 15) 6. The system of item 5, wherein the L protein is an L protein of encephalomyocarditis virus (EMCV) or a functional variant thereof. (Item 16) The system described in item 5, wherein the L protein shares at least 90% identity with SEQ ID NO:2. (Item 17) 6. The system of item 5, wherein the L protein is selected from the group consisting of a poliovirus L protein, an HRV16 L protein, a Mengovirus L protein, and a Saffold virus 2 L protein, or a functional variant thereof. (Item 18) 18. The system according to any one of items 1 to 17, wherein the system comprises a single vector comprising an expression cassette, the expression cassette comprising the first polynucleotide and the second polynucleotide. (Item 19) 19. The system of claim 18, 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 20) 19. The system of claim 18, wherein the expression cassette comprises a shared promoter operably linked to both the first polynucleotide and the second polynucleotide. (Item 21) 21. The system of claim 20, wherein the expression cassette comprises a coding polynucleotide comprising the first polynucleotide and the second polynucleotide linked by a polynucleotide encoding a ribosomal skipping site, the coding polynucleotide being operably linked to the shared promoter. (Item 22) 21. The system of claim 20, wherein the expression cassette comprises a coding polynucleotide, the coding polynucleotide encoding the enhancer protein and the target protein linked by a ribosomal skipping site, the coding polynucleotide being operably linked to the shared promoter. (Item 23) 23. The system of any one of items 18 to 22, wherein the expression cassette is configured for transcription of a single messenger RNA encoding both the target protein and the enhancer protein linked by a ribosomal skipping site, and translation of the messenger RNA results in expression of the target protein and the L protein as separate polypeptides. (Item 24) 24. The system according to any one of items 1 to 23, comprising one type of 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; and The system according to any one of items 1 to 17, comprising: (Item 26) The system according to any one of items 1 to 17 or 25, comprising two types of vectors. (Item 27) 27. The system according to any one of items 1 to 26, wherein either the first polynucleotide or the second polynucleotide, or both, are operably linked to an internal ribosome entry site (IRES). (Item 28) 28. The system of any one of items 1 to 27, wherein at least one of the one or more vectors comprises a T7 promoter configured for transcription of either or both of the first polynucleotide and the second polynucleotide by T7 RNA polymerase. (Item 29) 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 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; A vector, wherein the first polynucleotide and the second polynucleotide are operably linked to at least one promoter. (Item 31) 31. The vector of 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) 31. The vector of claim 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 expressing 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, wherein the exogenous polynucleotide is operably linked to a promoter. (Item 34) 34. The eukaryotic cell of claim 33, wherein the polynucleotide is operably linked to an internal ribosome entry site (IRES). (Item 35) 35. The eukaryotic cell of claim 33 or 34, wherein the promoter is an inducible promoter. (Item 36) 36. A method for recombinant expression of a target protein, comprising the step of introducing into a cell according to any one of items 33 to 35 a polynucleotide encoding said target protein, operably linked to a promoter. (Item 37) 33. 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) 38. The method of claim 36 or 37, wherein the target protein is a membrane protein. (Item 39) 39. The method of claim 38, wherein localization of the membrane protein to the cell membrane is increased compared to the localization observed when the membrane protein is expressed without the enhancer protein. (Item 40) A eukaryotic cell produced by introducing 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 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 42) 1. A method for expressing a target protein in a eukaryotic cell, comprising the steps of introducing into said eukaryotic cell a polynucleotide encoding said target protein, said polynucleotide being operably linked to a promoter; the method utilizing 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 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) 43. The method of claim 42, wherein the co-expression of an enhancer protein comprises introducing into the eukaryotic cell a polynucleotide encoding the enhancer protein operably linked to a promoter. (Item 44) 44. The method of claim 42 or 43, wherein the introducing step(s) comprises transfecting the eukaryotic cell with one or more DNA molecules, transducing the eukaryotic cell with a single viral vector, and / or transducing the eukaryotic cell with two viral vectors. (Item 45) 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 soluble protein. (Item 46) 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, wherein the target protein is a secreted protein. (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 to 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 to 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 comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 16. (Item 51) 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 ITK and, optionally, the ITK comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 15. (Item 52) 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 NADase, and optionally the NADase comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 20. (Item 53) A method for producing an antibody against a target protein, the method comprising a step of immunizing a subject with the cell of any one of items 33 to 35, the cell of item 40, or the target protein of item 41. (Item 54) 54. The method of claim 53, further comprising isolating one or more immune cells that express an immunoglobulin protein specific for the target protein. (Item 55) 55. The method of claim 53 or 54, comprising generating one or more hybridomas from the one or more immune cells. (Item 56) 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) 1. A method for antibody discovery by cell sorting comprising: 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; b) a population of recombinant cells, wherein the recombinant cells express a library of polypeptides, each of which comprises an antibody or antigen-binding fragment thereof; providing a solution comprising: and isolating one or more recombinant cells from said solution by sorting for said labeled cells or recombinant cells that are bound to said labeled target protein. (Item 58) 1. A method for panning a phage display library, comprising: a) mixing a phage display library with the eukaryotic 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; b) purifying and / or enriching members of the phage display library that bind to the cell or target protein; The method includes: (Item 59) 41. 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) 60. The eukaryotic cell according to any one of items 33 to 35, 40 and 59, which is a eukaryotic cell line. (Item 61) 61. The eukaryotic cell according to any one of items 33 to 35, 40, 59 and 60, which is a 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. (Item 62) 61. The eukaryotic cell of item 60, wherein the eukaryotic cell line is a stable cell line. (Item 63) 53. 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 an adeno-associated virus (AAV) vector, a lentivirus vector, a retrovirus vector, a replication-competent adenovirus vector, a replication-deficient adenovirus vector, a herpes virus vector, a baculovirus vector or a non-viral plasmid. (Item 64) 64. The system of claim 63, wherein at least one of the one or more vectors is an AAV vector. (Item 65) 33. 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 herpes virus vector, a baculovirus vector, or a non-viral plasmid. (Item 66) 66. The vector according to item 65, which is an AAV vector. (Item 67) The system of item 4, wherein the rhabdovirus matrix (M) protein is the M protein of vesicular stomatitis virus (VSV). (Item 68) 68. The system of claim 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, said one or more vectors comprising: a) a first polynucleotide encoding the target protein; b) a second polynucleotide encoding an L protein of an encephalomyocarditis virus (EMCV), optionally wherein said L protein shares at least 90% identity with SEQ ID NO:2; and 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, said one or more vectors comprising: a) a first polynucleotide encoding the target protein; b) a second polynucleotide encoding an L protein of Theiler's virus, optionally wherein said L protein shares at least 90% identity with SEQ ID NO:1; 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, said one or more vectors comprising: a) a first polynucleotide encoding the target protein; b) a second polynucleotide encoding a picornavirus 2A protease, optionally wherein said picornavirus 2A protease shares at least 90% identity with SEQ ID NO:7; and 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, said one or more vectors comprising: a) a first polynucleotide encoding the target protein; b) a second polynucleotide encoding an M protein of Vesicular Stomatitis Virus (VSV), optionally wherein said M protein shares at least 90% identity with SEQ ID NO:9; and wherein the first polynucleotide and the second polynucleotide are operably linked to one or more promoters. (Item 73) 73. The system according to any one of items 69 to 72, wherein the target protein is dopamine receptor 1 (DRD1), and optionally the DRD1 comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 19. (Item 74) 73. The system of any one of items 69 to 72, 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 to the amino acid sequence of SEQ ID NO: 18. (Item 75) 73. The system according to any one of items 69 to 72, wherein the target protein is C1 esterase inhibitor (C1-Inh), and optionally the C1-Inh comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 16. (Item 76) 73. The system according to any one of items 69 to 72, wherein the target protein is ITK, and optionally the ITK comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 15. (Item 77) 73. The system according to any one of items 69 to 72, wherein the target protein is an NADase, and optionally the NADase comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 20. [Brief description of the drawings]

[0010] [Figure 1]FIG. 1 depicts six exemplary modes of regulating gene expression in eukaryotic cells.

[0011] [Figure 2-1] Figures 2A-2Y are schematic diagrams of non-limiting exemplary constructs: EG1, Figure 2A; EG2, Figure 2B; EG3 and EG4, Figure 2C; EG5, Figure 2D; EG6, Figure 2E; EG7, Figure 2F; EG8, Figure 2G; EG9, Figure 2H; EG10 and EG11, Figure 2I; EG12 and EG4, Figure 2J; EG10, Figure 2K; EG13, Figure 2L; EG14, Figure 2M; EG15, Figure 2N; EG16, Figure 2O; EG17, Figure 2P; EG18, Figure 2Q; EG19, Figure 2R; EG20, Figure 2S; EG21, Figure 2T; EG22, Figure 2U; EG23, Figure 2V; EG24, Figure 2W; and EG25, Figure 2X. [Figure 2-2] Figures 2A-2Y are schematic diagrams of non-limiting exemplary constructs: EG1, Figure 2A; EG2, Figure 2B; EG3 and EG4, Figure 2C; EG5, Figure 2D; EG6, Figure 2E; EG7, Figure 2F; EG8, Figure 2G; EG9, Figure 2H; EG10 and EG11, Figure 2I; EG12 and EG4, Figure 2J; EG10, Figure 2K; EG13, Figure 2L; EG14, Figure 2M; EG15, Figure 2N; EG16, Figure 2O; EG17, Figure 2P; EG18, Figure 2Q; EG19, Figure 2R; EG20, Figure 2S; EG21, Figure 2T; EG22, Figure 2U; EG23, Figure 2V; EG24, Figure 2W; and EG25, Figure 2X. [Figure 2-3] Figures 2A-2Y are schematic diagrams of non-limiting exemplary constructs: EG1, Figure 2A; EG2, Figure 2B; EG3 and EG4, Figure 2C; EG5, Figure 2D; EG6, Figure 2E; EG7, Figure 2F; EG8, Figure 2G; EG9, Figure 2H; EG10 and EG11, Figure 2I; EG12 and EG4, Figure 2J; EG10, Figure 2K; EG13, Figure 2L; EG14, Figure 2M; EG15, Figure 2N; EG16, Figure 2O; EG17, Figure 2P; EG18, Figure 2Q; EG19, Figure 2R; EG20, Figure 2S; EG21, Figure 2T; EG22, Figure 2U; EG23, Figure 2V; EG24, Figure 2W; and EG25, Figure 2X.

[0012] [Diagram 3]Figures 3A-D show optical and fluorescent microscopy images of cells expressing GFP expressed using construct EG2 (CMV-GFP-IRES-L) compared to the control vector EG1. Figure 3A: Optical microscopy of cells containing EG1. Figure 3B: Fluorescence microscopy of cells containing EG1. Figure 3C: Optical microscopy of cells containing EG2. Figure 3D: Fluorescence microscopy of cells containing EG2. Expression of fluorescent GFP protein from the EG2 construct demonstrates the feasibility of the system. Reduction of deleterious overexpression in cells containing EG2 (Figure 3D) compared to cells containing EG1 (Figure 3B) demonstrates improved regulation of GFP expression by introduction of the L-protein. Bars in Figures 3A-D represent 400 microns.

[0013] [Figure 4] Figures 4A-D show optical and fluorescent microscopy images of cells expressing GFP expressed using constructs EG3 and EG4 (T7-IRES-L-GFP and CMV-T7, respectively) compared to the control vector EG1. Figure 4A: Optical microscopy of cells containing EG1. Figure 4B: Fluorescence microscopy of cells containing EG1. Figure 4C: Optical microscopy of cells containing EG3+EG4. Figure 4D: Fluorescence microscopy of cells containing EG3+EG4. Expression of fluorescent GFP protein from the EG3+EG4 construct demonstrates the feasibility of the system. The reduced expression in cells containing EG3+EG4 (Figure 4D) compared to cells containing EG1 (Figure 4B) demonstrates the improved regulation of GFP expression by introduction of the L-protein. Bars in Figures 4A-D represent 400 microns.

[0014] [Diagram 5]Figures 5A-D show fluorescence microscopy images of cells expressing DRD1-GFP fusions from constructs EG10 (CMV-[DRD1-GFP]) (Figure 5A) or EG8 (CMV-[DRD1-GFP]-IRES-L) (Figure 5C). DRD1-GFP using construct EG10 is expressed but is unable to transport the receptor into the outer membrane, resulting in the formation of inclusions (Figure 5B, arrows). DRD1-GFP using construct EG8 is expressed and robustly transported into the membrane, resulting in high yields of GPCR in the outer membrane with high quality (Figure 5D).

[0015] [Figure 6] Figures 6A-B show fluorescent microscopy images of cells expressing DRD1-GFP fusion protein expressed from constructs EG10 (CMV-[DRD1-GFP]) (Figure 6A) or EG12 and EG4 (T7-IRES-L-DRD1-GFP and CMV-T7, respectively) (Figure 6B). DRD1-GFP expressed using EG10 is expressed but fails to correctly transport the receptor into the outer membrane, resulting in the formation of inclusions (Figure 6A, arrows). DRD1-GFP expressed using EG12 in combination with EG4 is expressed and robustly transported into the membrane, resulting in high yields of GPCR in the outer membrane at high quality (Figure 6B).

[0016] [Figure 7] Figure 7 shows the results of an anti-CFTR Western blot. Co-expression of the L-protein and CFTR delivered as a PCR product or vector (left of dashed line) results in reduced yields but more homogenous samples compared to control expression of CFTR without co-expression of the L-protein (right of dashed line).

[0017] [Figure 8]Figures 8A-8B show the results of NADase purification and activity testing. Figure 8A shows SDS-PAGE of NADase affinity purified using the FLAG tag (standard, SeeBlue2 plus; lane 2, lysate / load; lane 3, flow-through; lane 4, column elution fraction 1; lane 5, column elution fraction 2; lane 6, column elution fraction 3; lane 7, column elution fraction 4; lane 8, resin). Figure 8B shows a graph of NAD+ conversion activity analyzed by high performance liquid chromatography (HPLC) using different concentrations of purified NADase.

[0018] [Figure 9] Figures 9A-B show the results of His-tag purification of ITK. Figure 9A shows SDS-PAGE of ITK affinity purified using His-tag. Lanes: lane 1, SeeBlue2 plus stained; lane 2, 500 ng GFP; lane 3, 2 μg ITK; lane 4, 5 μg ITK; lane 5, 10 μg ITK. Figure 9B shows Western blot analysis after SDS-PAGE of Figure 9A, with arrows pointing to ITK monomers and dimers.

[0019] [Figure 10] Figure 10 shows fluorescent microscopy images of cells expressing DRD1-GFP fusion protein expressed from constructs EG10(CMV-[DRD1-GFP]) (Figure 10A) or EG10 and EG11 (Figure 10B). The arrows point to inclusions formed by DRD1-GFP expressed from EG10, which is unable to properly transport the receptor into the outer membrane.

[0020] [Figure 11] Figure 11 shows a graph showing luminescence resulting from a cAMP-Glo™ assay, indicating cAMP levels in cells expressing either the E5 construct (CMV-DRD1-Strp) or the E6 construct (CMV-DRD1-Strp-IRES-L) in HEK293 cells in the presence or absence of dopamine. Higher luminescence signal indicates higher functional activity of DRD1-Strep.

[0021] [Figure 12] Figure 12 shows fluorescent microscopy images of cells expressing DRD1-GFP fusion protein expressed using the CMV promoter (Figure 12A), DRD1-GFP fusion protein expressed in combination with the L protein using the CMV promoter (Figure 12B), DRD1-GFP fusion protein expressed in combination with the L protein using the EF1-α promoter (Figure 12C), and DRD1-GFP fusion protein expressed in combination with the L protein using the SV40 promoter (Figure 12D). The lower panels show enlarged views of some of the cells shown in the upper panels.

[0022] [Figure 13] Figure 13 shows fluorescent microscopy images of HEK293 cells expressing DRD1-GFP fusion protein (Figure 13A), DRD1-GFP fusion protein expressed in combination with the L protein from EMCV (Figure 13B), DRD1-GFP fusion protein expressed in combination with the L protein from Theiler's virus (Figure 13C), DRD1-GFP fusion protein expressed in combination with the 2A protease of poliovirus (Figure 13D), and DRD1-GFP fusion protein expressed in combination with the M protein of vesicular stomatitis virus (Figure 13E). The lower panels show enlarged views of some of the cells shown in the upper panels.

[0023] [Figure 14] Figure 14 shows fluorescent microscopy images of CHO-K1 cells expressing DRD1-GFP fusion protein (Figure 14A) and DRD1-GFP fusion protein expressed in combination with the L protein from Theiler's virus (Figure 14B). The arrows point to inclusions formed by DRD1-GFP expressed from EG10, which is unable to properly transport the receptor into the outer membrane.

[0024] [Figure 15]Figure 15 shows fluorescent microscopy images of CHO-K1 cells expressing DRD1-GFP fusion protein (Figure 15A) and DRD1-GFP fusion protein expressed in combination with the L protein from EMCV (Figure 15B). In Figure 15A, the arrow points to the inclusions formed by DRD1-GFP expressed from EG10, which is unable to correctly transport the receptor into the outer membrane. In Figure 15B, the arrow points to DRD1-GFP that is correctly localized and incorporated into the membrane.

[0025] [Figure 16-1] Figure 16 shows images of SDS-PAGE analysis of ITK protein expressed in HEK293 cells purified using nickel-loaded affinity resin (Figure 16A) or size-exclusion chromatography (Figure 16B), and ITK-L fusion protein expressed in HEK293 cells purified using nickel-loaded affinity resin (Figure 16C) or size-exclusion chromatography (Figure 16D). P1 refers to the dimeric form of ITK, while P2 refers to the monomeric form of ITK. [Figure 16-2] Figure 16 shows images of SDS-PAGE analysis of ITK protein expressed in HEK293 cells purified using nickel-loaded affinity resin (Figure 16A) or size-exclusion chromatography (Figure 16B), and ITK-L fusion protein expressed in HEK293 cells purified using nickel-loaded affinity resin (Figure 16C) or size-exclusion chromatography (Figure 16D). P1 refers to the dimeric form of ITK, while P2 refers to the monomeric form of ITK.

[0026] [Figure 17-1] Figure 17A shows the results of SDS-PAGE analysis of purified ITK protein and purified ITK protein expressed in combination with L protein in HEK293 cells, and Figure 17B shows a graph of luminescence measurements of P1 and P2 ITK purified protein samples as indicated on the SDS PAGE image. [Figure 17-2]Figure 17A shows the results of SDS-PAGE analysis of purified ITK protein and purified ITK protein expressed in combination with L protein in HEK293 cells, and Figure 17B shows a graph of luminescence measurements of P1 and P2 ITK purified protein samples as indicated on the SDS PAGE image.

[0027] [Figure 18-1] Figure 18 shows images of SDS-PAGE analysis of ITK protein expressed in CHO cells purified using nickel-loaded affinity resin (Figure 18A) or size-exclusion chromatography (Figure 18B) and ITK protein expressed in combination with L protein in CHO cells purified using nickel-loaded affinity resin (Figure 18C) or size-exclusion chromatography (Figure 18D). P1 refers to the dimeric form of ITK, while P2 refers to the monomeric form of ITK. [Figure 18-2] Figure 18 shows images of SDS-PAGE analysis of ITK protein expressed in CHO cells purified using nickel-loaded affinity resin (Figure 18A) or size-exclusion chromatography (Figure 18B) and ITK protein expressed in combination with L protein in CHO cells purified using nickel-loaded affinity resin (Figure 18C) or size-exclusion chromatography (Figure 18D). P1 refers to the dimeric form of ITK, while P2 refers to the monomeric form of ITK. [Figure 18-3] Figure 18 shows images of SDS-PAGE analysis of ITK protein expressed in CHO cells purified using nickel-loaded affinity resin (Figure 18A) or size-exclusion chromatography (Figure 18B) and ITK protein expressed in combination with L protein in CHO cells purified using nickel-loaded affinity resin (Figure 18C) or size-exclusion chromatography (Figure 18D). P1 refers to the dimeric form of ITK, while P2 refers to the monomeric form of ITK. [Figure 18-4]Figure 18 shows images of SDS-PAGE analysis of ITK protein expressed in CHO cells purified using nickel-loaded affinity resin (Figure 18A) or size-exclusion chromatography (Figure 18B) and ITK protein expressed in combination with L protein in CHO cells purified using nickel-loaded affinity resin (Figure 18C) or size-exclusion chromatography (Figure 18D). P1 refers to the dimeric form of ITK, while P2 refers to the monomeric form of ITK.

[0028] [Figure 19] FIG. 19 shows a graph of luminescence measurements of P1 and P2 ITK protein samples expressed in combination with the L protein in CHO cells and purified using a size exclusion chromatography experiment.

[0029] [Figure 20] Figure 20A shows an image of an SDS-PAGE analysis of purified C1-inhibitor expressed in the absence (left) or presence (right) of Enhancer L protein. Figure 20B shows a graph depicting the concentration of functionally active C1-inhibitor present in purified C1-inhibitor protein samples expressed in the presence or absence of Enhancer L protein, as indicated. Data was obtained using a commercially available Quidel MicroVue Complement C1-Inhibitor Plus enzyme immunoassay, using C1-inhibitor-containing healthy human plasma as a positive control (100% activity) as per the manufacturer's protocol.

[0030] [Figure 21-1] Figure 21 shows ion exchange chromatography of PSG1. Protein-containing fractions (Figure 21A, red box) were pooled and concentrated before confirming the presence of PSG1 by SDS-PAGE and Western blot (Figure 21B, red arrow). [Figure 21-2]Figure 21 shows ion exchange chromatography of PSG1. Protein-containing fractions (Figure 21A, red box) were pooled and concentrated before confirming the presence of PSG1 by SDS-PAGE and Western blot (Figure 21B, red arrow). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Detailed Description In the present disclosure, a vector system, vector or eukaryotic cell is provided that is useful for co-expression of enhancer protein and target protein. In some embodiments, a system for recombinant expression of target protein in eukaryotic cell is provided, comprising one or more vectors. In some embodiments, the vector(s) has a first polynucleotide that encodes a target protein and a second polynucleotide that encodes 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, it is believed that the compositions and methods of the present disclosure prevent cellular regulatory mechanisms from activating in response to the expression of recombinant target proteins, thereby improving the yield and / or functionality of the target proteins. 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 Figures 2A-2Y and Table 1. In some embodiments, a first vector includes a polynucleotide encoding a target protein and a second vector includes a polynucleotide encoding an enhancer protein. In other embodiments, a single vector includes one or more polynucleotides encoding a target protein and an enhancer protein. A vector can include a single polynucleotide encoding both a target protein and an enhancer protein. In the alternative, more than one enhancer protein and / or more than one target protein are encoded by the vector(s).

[0034] Polynucleotides The present disclosure relates to recombinant polynucleotides for the expression of one or more target proteins and one or more enhancer proteins. The polynucleotides (or nucleic acids or nucleic acid molecules) can include one or more genes of interest and are 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 polynucleotides are isolated from natural sources; 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 polynucleotides are of any shape (linear, circular, etc.) or topology (single-stranded, double-stranded, linear, circular, supercoiled, twisted, nicked, etc.). Polynucleotides 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; and nucleic acids having chemically modified 5' or 3' termini; and nucleic acids having two or more of such modifications. All linkages in a polynucleotide need not be identical.

[0035] Examples of polynucleotides include, without limitation, oligonucleotides (including, but not limited to, antisense oligonucleotides, ribozymes, and oligonucleotides useful in 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 an in vitro transcribed (IVT) mRNA. In some embodiments, the polynucleotide is a plasmid.

[0036] A polynucleotide is said to "encode" a protein if it comprises 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 performed by endogenous or exogenous enzymes. In some embodiments, transcription of the polynucleotides of the present disclosure is performed by the endogenous polymerase II (polII) of the eukaryotic cell. In some embodiments, an 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" that encodes a target protein; a "second polynucleotide" that encodes an enhancer protein; and a "coding polynucleotide" that encodes one or more target proteins, one or more enhancer proteins, and / or one or more separation elements.

[0038] Target Protein The polynucleotides of the present disclosure can include nucleic acid sequences encoding one or more target proteins. The nucleic acid sequences encoding the target proteins are referred to as 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 conjunction 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 that includes one or more mutations in the native AAV capsid protein sequence.

[0039] Target proteins for expression using the present compositions and methods can include proteins involved in 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 present 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 present compositions and methods are used for the production of therapeutic proteins. In some embodiments, the protein is a human protein and the host cell for expression is a human cell.

[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, ancestim, anistreplase, anthrax immunoglobulin human, antihemophilic factor, antithrombin alpha, antithrombin III human, antithymocyte globulin, antithymocyte globulin (horse), antithymocyte globulin (rabbit), aprotinin, arcitumomab, asfotase alpha, 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, sertoli Zumab pegol, cetuximab, choriogonadotropin alfa, chorionic gonadotropin (human), chorionic gonadotropin, coagulation factor IX, coagulation factor VIIa, coagulation factor X human, coagulation factor XIII A subunit, collagenase, conestat alfa, corticotropin, cosyntropin, daclizumab, daptomycin, daratumumab, darbepoetin alfa, defibrotide, denileukin diftitoc s, denosumab, desirudin, dinutuximab, dornase alfa, drotrecogin alfa, dulaglutide, eculizumab, efalizumab, efmoroctocog alfa, elosulfase alfa, elotuzumab, enfuvirtide, epoetin alfa, epoetin zeta, eptifibatide, etanercept, evolocumab, exenatide, factor IX complex (human), concentrated Fibrinogen (human), Fibrinolysin aka Plasmin, Filgrastim, Filgrastim-sndz, Follitropin alfa, Follitropin beta, Galsulfase, Intrinsic gastric factor, Gemtuzumab ozogamicin, Glatiramer acetate, Glucagon recombinant, Glucarpidase, Golimumab, Gramicidin D, Hepatitis A vaccine, Hepatitis B immunoglobulin, Human calcitonin, Human Clostridiumtetani 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 alfa-2a, recombinant, interferon alfa-2b, interferon alfacon-1, interferon alfa-n1, interferon alfa-n9, interferon beta-1a, interferon beta-1b, interferon gamma-1b, intravenous immunoglobulin, ipilimumab Mumab, Ixekizumab, Laronidase, Lenograstim, Lepirudin, Leuprolide, Liraglutide, Lucinactant, Lutropin alfa, Lutropin alfa, Mecasermin, Menotropin, Mepolizumab, Epoetin beta, Metreleptin, Muromonab, Natalizumab, Alpha interferon, Necitumumab, Nesiritide, Nivolumab, Obiltoxaximab, Obinutuzumab, Ocriplasmin, Ofatumumab, Omalizumab, Opregnat Rubekin, OspA lipoprotein, Oxytocin, Palifermin, Palivizumab, Pancrelipase, Panitumumab, Pembrolizumab, Pertuzumab, Boractant alfa, Pramlintide, Preotact, Protein S human, Ramucirumab, Ranibizumab, Rasburicase, Raxibacumab, Reteplase, Rilonacept, Rituximab, Romiplostim, Sacrosidase, Salmon calcitonin, Sargramostim, Satumomab pendetidePendetide), Sebelipase alfa, Secretin, Secukinumab, Sermorelin, Serum Albumin, Iodized Serum Albumin, Siltuximab, Simoctocog alfa, Sipuleucel-T, Somatotropin Recombinant, Somatropin Recombinant, Streptokinase, Sulodexide, Susoctocog alfa, Taliglucerase alfa, Teduglutide, Teicoplanin, Tenecteplase, Teriparatide, Tesamorelin, Thrombomodulin alfa, Thymalfasin, Thyroglobulin, Thyrotropin alpha, Thyrotropin alpha, Tocilizumab, Tositumomab, Trastuzumab, Tuberculin Purified Protein Derivative, Turoctocog alfa, Urofollitropin, Urokinase, Ustekinumab, Vasopressin, Vedolizumab, and Velaglucerase alfa.

[0041] In some embodiments, the target protein is, but is not limited to, soluble protein, secreted protein or 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, the SARM1 is a deletion variant that represents the mature protein.

[0042] In some embodiments, the target protein is a membrane protein. Exemplary membrane proteins include ion channels, gap junctions, ionotropic 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 signal transduction (e.g., Ras, Rac, Raf, Gα subunits, arrestins, Src and other effector proteins), etc. In some embodiments, the membrane protein is a G protein-coupled receptor. In some embodiments, the target protein is a 7 (transmembrane) domain receptor, a 7TM receptor, a heptahelical receptor, a serpentine receptor, or a G protein-linked receptor (GPLR). In some embodiments, the target protein is a class A GPCR, a class B GPCR, a class C GPCR, a class D GPCR, a class E GPCR, or a 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, a Secretin Receptor Family GPCR, a Metabotropic Glutamate / Pheromone GPCR, a Fungal Mating Pheromone Receptor, a Cyclic AMP Receptor, or a Frizzled / Smoothened GPCR.

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

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

[0045] Enhancer Proteins The present disclosure relates to the co-expression of target protein and enhancer protein.In some embodiments, 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 can reduce one or more of misfolding, altered activity, incorrect post-translational modification and / or toxicity.

[0046] In some embodiments, the enhancer protein is a nuclear pore blocking viral protein. In some embodiments, the enhancer protein is a native or synthetic peptide that can block nuclear pore, thereby inhibiting nucleocytoplasmic transport ("NCT"). In some embodiments, the enhancer protein is a viral protein. In some aspects, the viral 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] Enhancer protein is any functional variant of the proteins disclosed herein.As used herein, the term "functional variant" refers to a protein that is homologous to and / or shares substantial sequence similarity with the original protein (e.g., more 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 from a picornavirus or a functional variant thereof. In some embodiments, the enhancer protein is a leader protein from the Cardiovirus, Hepatovirus or Aphthovirus genus. For example, the enhancer protein can be from Bovine Rhinitis A virus, Bovine Rhinitis B virus, Equine Rhinitis A virus, Foot and Mouth Disease virus, Hepatovirus A, Hepatovirus B, Marmota himalayana hepatovirus, 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 an encephalomyocarditis virus (EMCV) L protein 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 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 a poliovirus L protein, an HRV16 L protein, a Mengovirus L protein, and a Saffold virus 2 L protein, or a functional variant thereof.

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

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

[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 nuclear import complex formation and / or disrupts STAT1 transport into the nucleus.

[0056] In some embodiments, the enhancer protein is Ebola virus VP24 protein or a functional variant thereof. In some embodiments, the enhancer protein is Ebola virus VP40 protein or VP35 protein. In some embodiments, the enhancer protein is a viral protein that binds to the importin protein karyopherin-alpha (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 a herpes simplex virus (HSV) ICP27 protein or a functional variant thereof. In some embodiments, the enhancer protein is an HSV ORF57 protein.

[0059] In some embodiments, the enhancer protein is rhabdovirus matrix (M) protein or its functional variant.In some embodiments, the enhancer protein is the M protein from Cytorhabdovirus, Dichoravirus, Ephemerovirus, Lyssavirus, Novirhabdovirus, Nucleorhabdovirus, Perhabdovirus, Sigmavirus, Spibivirus, 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 an amino acid sequence 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 an amino acid sequence 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 an amino acid sequence 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 that comprises, consists of, or consists essentially of one of the amino acid sequences listed in Table 1. In some embodiments, the enhancer protein can have an amino acid sequence that comprises, consists of, or consists 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 proteins In some embodiments, the target protein and the enhancer protein are contained in a single fusion protein. In some embodiments, the fusion protein can include a linking element. In some embodiments, the linking element can include a cleavage site for enzymatic cleavage. In other embodiments, the fusion protein or linking 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 polynucleotides 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 allow its detection, such as a radiolabel, a fluorescent agent, biotin, a peptide tag, an enzyme fragment, etc. The proteins may include affinity tags, such as His-tags, FLAG tags, GST-tags, Strep-tags, biotin-tags, immunoglobulin binding domains, such as IgG binding domains, calmodulin binding peptides, etc. 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 selectable markers, such as antibiotic resistance markers.

[0063] Polymerase Endogenous or exogenous polymerases can be used for transcription of the polynucleotides encoding the target protein(s) and enhancer protein(s). In some embodiments, transcription of the polynucleotide(s) is performed by a natural polymerase contained by the cell (e.g., a eukaryotic cell). Viral polymerases can be used instead or in addition. In some embodiments, viral promoters are used in combination with one or more viral polymerases. In some embodiments, eukaryotic promoters are 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) of the present disclosure may comprise a polynucleotide sequence encoding a polymerase. In some embodiments, the polymerase is a viral polymerase. The polynucleotide sequence encoding the polymerase may be comprised by a vector that comprises a target protein-encoding polynucleotide and / or an enhancer protein-encoding polynucleotide. In some embodiments, the polymerase may be comprised by a vector that does not comprise a target protein- or enhancer protein-encoding polynucleotide.

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

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

[0067] The vector 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, such as 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. In some embodiments, the vector is a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage, or an artificial chromosome. In some embodiments, the vector is a viral vector, including an adenovirus vector, a retrovirus vector, or an adeno-associated virus vector. In some embodiments, the vector is a bacterial artificial chromosome (BAC), a plasmid, a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome (YAC), or a 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 that encodes a target protein and a second polynucleotide that encodes 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 comprising a first polynucleotide operably linked to a first promoter, and a second vector comprising a second polynucleotide operably linked to a second promoter.

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

[0071] In some embodiments, a cell is provided that comprises the vector(s) of the present disclosure. In some embodiments, a cell is provided that comprises the polynucleotide of the present disclosure. In some embodiments, a cell is provided that expresses the target protein(s) and enhancer protein(s) of the present disclosure.

[0072] promoter The vectors of 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 initiation site that is involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. The polynucleotide(s) or vector(s) of 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, the one or more promoters are native promoters. In some embodiments, the one or more promoters are non-native promoters. In some embodiments, the 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, splenic 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 their ability to operate, not by their physical location. The elements or structures are capable of or are characterized by the accomplishment of a desired operation. It will be appreciated by those skilled in the art that elements or structures in a nucleic acid sequence need not be in tandem or contiguous order to be operably linked.

[0074] In some embodiments, the promoter constitutively drives expression of one or more target proteins and / or one or more enhancer proteins; i.e., the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. The inducible promoter can be, but is not limited to, any inducible promoter known in the art. 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 transcription 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 comprises 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 comprises 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 the transcription factor.In some embodiments, the transcription factor is bacterial EL222, which dimerizes in the presence of blue light and drives the expression from the C120 promoter or its regulatory element.In some embodiments, the inducible promoter comprises the nucleic acid sequence derived from the C120 promoter or regulatory element.

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

[0078] Expression cassette The vector(s) of the present disclosure can include one or more expression cassettes. The phrase "expression cassette" as used herein 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, the expression cassette including 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 separation element (e.g., a ribosomal skipping site or a 2A element), and the coding polynucleotides are operably linked to a shared promoter.

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

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

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

[0083] In some embodiments, the expression cassette is an adeno-associated virus (AAV) expression cassette that includes a 5' inverted terminal repeat (ITR), any one of the nucleic acid sequences disclosed herein for expression of one or more target proteins and one or more enhancer proteins, and a 3' ITR. In some embodiments, the AAV expression cassette includes 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 the nucleic acid sequences for one or more target proteins and one or more enhancer proteins are carried by the same vector, the vector can include a separation element 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 viral (AAV) vector, a lentiviral vector, a retroviral vector, a replication-competent adenoviral vector, a replication-defective adenoviral vector, a herpes virus vector or a baculovirus vector.

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

[0089] In some embodiments, the method includes contacting the AAV producer cell with one or more additional plasmids, e.g., containing the AAV rep and cap genes and / or encoding helper virus protein sequences, hi some embodiments, the method further includes maintaining the AAV producer cell under conditions such that AAV is produced.

[0090] The present disclosure provides a rAAV vector produced using any one of the methods disclosed herein. The produced rAAV vector can be of any serotype, 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 include one or more amino acid modifications (e.g., substitutions and / or deletions) compared to native AAV capsid. In some embodiments, the recombinant AAV vector is a single-stranded AAV (ssAAV). In some embodiments, the recombinant AAV vector is a self-complementary AAV (scAAV).

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

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

[0093] In some embodiments, the target protein is derived from a pathogen. The pathogen can be a virus, a bacterium, a fungus, or a 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 papilloma virus. 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 bacteria 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 a 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. The polynucleotides or vectors can be introduced into a cell by a variety of methods that are well known in the art and are selected, in part, based on the particular host cell. For example, the polynucleotides can be introduced into a cell using chemical, physical, biological, or viral means. Methods of introducing a polynucleotide or vector into a cell include, but are not limited to, the use of calcium phosphate, dendrimers, cationic polymers, lipofection, fugenes, peptide dendrimers, electroporation, cell squeezing, sonoporation, optical transfection, protoplast fusion, impalefection, hydrodynamic delivery, gene guns, magnetofection, microprojectile guns, nucleofection, and viral transduction.

[0095] The vector containing the targeting DNA and / or nucleic acid encoding the target protein and the enhancer protein can be introduced into the cell by various methods (e.g., injection, transformation, transfection, direct uptake, biolistic projectile, liposome). The target protein and the enhancer protein can be stably or transiently expressed in the cell 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, the polynucleotide or vector can be introduced into the host cell by insertion into the genome, using standard methods for producing stable cell lines, such as lentiviral transfection, baculovirus gene transfer into mammalian cells (BacMam), retroviral transfection, CRISPR / Cas9 and / or the use of transposons, as needed. In some embodiments, the polynucleotide or vector can be introduced into the host cell for transient transfection. In some embodiments, the transient transfection can be effected by the use of viral vectors, helper lipids, such as PEI, lipofectamine and / or Fectamine 293. The genetic elements can be encoded, for example, as DNA in a vector, or as RNA, for example, derived from PCR. The genetic elements can be separated in different vectors or combined in the same vector.

[0097] cells, cell lines, host cells Another aspect of the present 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 may be any of those described herein. The present 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 "super producer cells" or "super producer cell lines". In some embodiments, the super producer cells further comprise a polynucleotide and / or vector encoding one or more target proteins. Without being bound to any one theory, it is believed that a cell expressing one or more enhancer proteins disclosed herein can function as a host cell 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 post-translational modifications ("PTMs"), such as, for example, glycosylation, phosphorylation, disulfide bonds, etc., in the target protein. In some embodiments, a 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 cells are transiently transfected with any one or more of the polynucleotides and / or vectors disclosed herein.

[0101] In some embodiments, the present disclosure provides a eukaryotic cell for expression of a target protein, the cell comprising an exogenous polynucleotide encoding an enhancer protein. In some embodiments, the exogenous polynucleotide encoding the enhancer protein is transiently transduced and / or is not integrated into the genome of the cell. 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 (optionally a native promoter or an exogenous promoter). In some embodiments, the polynucleotide is operably linked to an internal ribosome entry site (IRES).

[0102] Protein Expression Methods The present disclosure provides a method for expressing a target protein in a eukaryotic cell. The method can include introducing a polynucleotide (operably linked to a promoter) encoding the target protein into a eukaryotic cell. The method utilizes the 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 the 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 one or more enhancers.In some embodiments, the expression level of the target protein expressed in combination with one or more enhancers according to the method of the present disclosure is at least about 1.1 times higher (e.g., about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times or about 10 times) compared to the expression level of the target protein expressed in the absence of one or more enhancers.

[0104] In some embodiments, the activity of the 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 one or more enhancers.In some embodiments, the activity of the target protein expressed in combination with one or more enhancers according to the method of the present disclosure is at least about 1.1 times higher (e.g., about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times or about 10 times) than the activity of the target protein expressed in the absence of 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 aspects, 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 carried out in a eukaryotic cell comprising one or more vectors. In some embodiments, the method is carried out using the polynucleotides, vectors and cells described in the previous section. In some embodiments, the vector(s) can have a first polynucleotide encoding the target protein and a second polynucleotide encoding the enhancer protein. In some embodiments, the first polynucleotide and the second polynucleotide are operably linked to one or more promoters.

[0107] Further provided is a method for recombinant expression of a target protein, comprising introducing a polynucleotide encoding the target protein into a eukaryotic cell, the polynucleotide being operably linked to a promoter. In some embodiments, the method of target protein expression 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 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 a membrane-associated membrane protein expressed in the absence of the one or more enhancers.

[0108] In some embodiments, expression of one or more enhancer proteins disclosed herein using the methods disclosed herein can be associated with, correlated with, or result in an effect on the cell cycle of a host cell, such as altering the number of enhancer-expressing host cells in a specific cell cycle stage compared to wild-type cells that do not express the one or more enhancer proteins. In some embodiments, expression of one or more enhancer proteins disclosed herein using the methods disclosed herein can be associated with, correlated with, or result in arrest of a host cell in a specific stage of the cell cycle. In some embodiments, the specific cell stage is a proliferative phase of the cell cycle, such as G1, S, or G2 phase. In some embodiments, expression of one or more enhancer proteins disclosed herein using the methods disclosed herein can be associated with, correlated with, or result in a reduction or elimination of clonal drift in a 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 transfecting the eukaryotic cell with one or more DNA molecules, transducing the eukaryotic cell with a single viral vector, and / or transducing the eukaryotic cell with two or more viral vectors.

[0110] Downstream Application In some embodiments, target proteins produced by the use of the compositions, systems and methods and 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, etc. In some embodiments, the compositions and methods are used to produce antibodies or produce antigens for antibody screening assays. In some embodiments, cells expressing target proteins can be used as assay systems, for example, to screen cell interactions, antibody binding, or small molecule effects in whole cell systems.

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

[0112] The present disclosure further provides a method for antibody discovery by cell sorting, comprising the steps of providing a solution containing a population of labeled cells or target proteins and recombinant cells produced using the disclosed system or method, wherein the recombinant cells express a library of polypeptides comprising antibodies or antigen-binding fragments thereof, respectively, and sorting one or more recombinant cells from the solution by detecting the labeled cells or recombinant cells bound to the labeled target proteins. In other variations, cell sorting is performed on cells derived from an immunized subject. The subject can be immunized with cells or target proteins produced according to the disclosed method, or with another suitable immunogen. In some embodiments, the recombinant cells contain a naive antibody library, optionally a human naive antibody library. A variety of antibody library generation methods are known in the art and can be combined with the disclosed method. As used herein, the term "sorting" or "cell sorting" refers to fluorescence-activated cell sorting, magnetic-assisted 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, the method comprising mixing the phage display library with a cell or target protein produced using a 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 (scFv) or other types of antibodies / antibody fragments (such as Fabs).

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

[0115] The ability to express antigens in their native or disease-relevant form, in high yield and / or on the surface of cells, allows for more reliable discovery and / or production than prior art methods of antibodies, antibody fragments and other molecules that may be useful as therapeutics and / or research tools, or for other applications.

[0116] In some embodiments, the disclosed systems and methods are suitable for use in discovering antibodies that bind and / or are specific for a particular glycosylation pattern in a target molecule (e.g., a glycoprotein). In some embodiments, an antibody library is selected against natively glycosylated proteins and counter-selected against improperly glycosylated or deglycosylated cognate proteins. Similarly described, by using deglycosylation enzymes, antibodies can be specifically selected against glycosylation patterns. In further embodiments, the disclosed cells and / or target proteins are used to validate the binding and / or functional activity of novel antibodies or other macromolecules.

[0117] In some embodiments, the disclosed system and method 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 disclosed system and method are suitable for use in the biosynthesis of any target protein in mammalian cells or using fermentation in bacteria, yeast and other microorganisms.In some embodiments, the disclosed system and method are suitable for use in the biosynthesis of non-protein molecules by introducing specific metabolic pathways into host cells.For example, the non-protein molecule is an opioid molecule or another metabolite.

[0118] Exemplary Benefits The present composition, system and method can have many advantages.For example, as demonstrated in Example 11, human NADase, which normally leads to apoptosis when overexpressed in human cell lines and thus produces undetectable yields, can be reliably expressed and produce yields of over 20mg / L when an enhancer protein is co-expressed with the target protein.Moreover, the NADase expressed by this exemplary method is functional (as demonstrated by phosphate release assay) and shows low batch-to-batch variation.

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

[0120] The cell of the present disclosure can have one or more of the following advantages in terms of target protein expression: higher concentration of target membrane protein in membrane; slower / reduced target protein degradation; improved signal-to-noise ratio in lysate; target protein and / or enhancer protein expression without affecting downstream cell metabolism; increased stability against desensitization of membrane-bound membrane protein; and higher target protein yield. Example 1 provides an illustrative example of enhancer protein expression without affecting downstream metabolism of cells. The GPCR illustrated in Example 1 can interact with its natural substrate and produce activation that can be measured in vitro.

[0121] The present systems and methods, in some embodiments, 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 difficult to express proteins.

[0122] system One aspect of the present disclosure provides a system for recombinant expression of target protein in eukaryotic cells, comprising one or more vectors. The vector(s) can have a first polynucleotide encoding the 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 rhabdovirus 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 nucleocytoplasmic 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 may be a picornavirus leader (L) protein or a functional variant thereof. In some embodiments, the NCT inhibitor may be a picornavirus 2A protease or a functional variant thereof. In some embodiments, the NCT inhibitor may be a rhinovirus 3C protease or a functional variant thereof. In some embodiments, the NCT inhibitor may be a coronavirus ORF6 protein or a functional variant thereof. In some embodiments, the NCT inhibitor may be an Ebola virus VP24 protein or a functional variant thereof. In some embodiments, the NCT inhibitor may 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 the L protein of Theiler's 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 an Encephalomyocarditis Virus (EMCV) L protein 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 a poliovirus L protein, an HRV16 L protein, a Mengovirus L protein, and a Saffold virus 2 L protein, or a functional variant thereof.

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

[0130] In some embodiments, the expression cassette comprises a coding polynucleotide comprising a first polynucleotide and a second polynucleotide linked by a polynucleotide encoding a ribosomal skipping site, wherein the coding polynucleotide is operably linked to a shared promoter.

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

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

[0133] The system can include one vector. In some embodiments, the system can include a single vector that includes a first polynucleotide that encodes a target protein and a second polynucleotide that encodes an enhancer protein.

[0134] The system can include two vectors, in some embodiments, the system can include a first vector comprising a first polynucleotide operably linked to a first promoter, and a second vector comprising 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 comprised by the system can include a T7 promoter configured for transcription of either the first polynucleotide or the second polynucleotide, or both, by T7 RNA polymerase.

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

[0138] All articles, publications, and patents cited herein are incorporated by reference as if each individual article, publication, or patent was specifically and individually indicated to be incorporated herein by reference, and are incorporated herein by reference as if they disclosed and described the methods and / or materials in connection with which the publications are cited. However, mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be taken as, an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

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

[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 belongs. EXAMPLES

[0141] [Table 4] Materials and Methods Construction of DNA molecules The entire assembly was made into a plasmid backbone that can be propagated in E. coli, containing a promoter (ColE1) controlling a high copy number origin of replication followed by terminators (rrnB T1 and T2 terminators). This is followed by a promoter controlling an antibiotic resistance gene, separated from the rest of the vector by a second terminator (a transcription terminator from phage lambda). The gene containing the backbone elements was synthesized by phosphoramidite chemistry.

[0142] The structural genes used in the construction of the plasmids 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 using the primers listed in Table 2. Selected amino acid sequences contained by 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 lines - culture and transfection HEK293 cells were used to illustrate the application of the system, method and composition in human eukaryotic cells. HEK293 adherent cells (CLS) were cultured in Dulbecco's Modified Eagle Medium High Glucose (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 50,000U Pen Strep (Gibco). HEK293 cells were grown to 80% confluence at 37°C and 5% CO2 before being transiently transfected using 293fectin (ThermoFisher) according to the manufacturer's instructions. Protein-expressing cells were harvested after 48 hours by detaching and scraping the cells using 0.5% trypsin solution for 5 minutes at 37°C. The cells were pelleted (5,000 x g, 15 minutes, 4°C) and the supernatant was discarded. The cell pellets were stored at -80°C until further use.

[0144] Suspension HEK293 cells were used to illustrate the application of the present system, method and composition in human eukaryotic cells. Suspension adapted HEK293 cells (CLS) were cultured in supplemented Expi293 expression medium (Gibco). One day prior to transfection, 1.75×10 6 Cells were seeded at 1000 cells / ml and incubated overnight at 37°C and 5% CO2, then transiently transfected using the Expi293 Expression System Kit (Gibco) according to the manufacturer's instructions. Protein-expressing cells were harvested after 48-96 h by centrifugation (5,000 x g, 15 min, 4°C). 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] CHO-K1 cells are used to illustrate the application of the system, method and composition in eukaryotic cells. CHO-K1 adherent cells (CLS) were cultured in DMEM / F-12 GlutaMAX medium (Gibco) supplemented with 10% fetal bovine serum (Gibco). CHO-K1 cells were grown to 80% confluence at 37°C and 5% CO2 before being transiently transfected using Lipofectamine LTX (ThermoFisher) according to the manufacturer's instructions. Protein-expressing cells were harvested after 48 hours by detaching and scraping the cells using 0.5% trypsin solution for 5 minutes at 37°C. The cells were pelleted (5,000 x g, 15 minutes, 4°C) and the supernatant was discarded. The cell pellets were stored at -80°C until further use.

[0146] SF9 cells were used to illustrate the application of the system, method and composition in eukaryotic insect cells. SF9 suspension cells (CLS) were cultured in Sf9-900 III medium (Gibco). SF9 cells were grown at 26°C and 130 rpm before seeding in 6-well plates for transient transfection using Cellfectin II (ThermoFisher) according to the manufacturer's instructions. Protein-expressing cells were harvested after 48 hours by detachment and pelleting (5,000 x g, 15 min, 4°C) and the supernatant was discarded. Cell pellets were stored at -80°C until further use.

[0147] Example 1 GFP expression in HEK293 cells CMV promoter system To demonstrate the impact of introducing viral nuclear pore blocking proteins on expression, HEK293 cells were transfected with either EG1, EG2 or co-transfected with EG3 and EG4 constructs (see Table 2 and Figure 2 for construct details). Expression of viral pore blocking proteins resulted in a controlled regulation of protein expression. As a result, the resulting GFP signal was reduced. The reason for the controlled regulation of the gene of interest in concert with the pore blocking proteins is the mechanism of action of the viral proteins. Without being bound by theory, a possible mechanism of protein regulation is that by expressing the pore blocking proteins, the nuclear export of mRNA can be inhibited, resulting in downregulation of the translation of the target protein. After stabilization, the pore blocking proteins will be degraded and mRNA transport will resume. This again results in the expression of both the target protein and the enhancer protein, e.g., the pore blocking protein. This tightly controlled feedback ensures the stabilization and permanent expression of the target protein, preventing the normal regulation of eukaryotic cells that leads to the cessation of protein expression.

[0148] 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. HEK293 cells were cultured in 24-well plates at 0.05×10 GFP expression cells prior to transient transfection with either EG1 or EG2 as described above. 6 Cells were seeded at 1000 cells / well and incubated overnight at 37° C. and 5% CO2. GFP expression was monitored after 24 and 48 hours using a fluorescent microscope. Images were taken using a CCD camera (Amscope) and analyzed with ISCapture (Amscope). This example demonstrates 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 natural polymerase of the eukaryotic host, but can use other viral polymerases, such as T7, to initiate transcription outside the nucleus. The viral polymerase is under the control of a standard eukaryotic promoter, and the corresponding mRNA will be dependent on nuclear export. In the cytosol, the viral polymerase is translated, which then initiates the transcription of the target protein polynucleotide and the enhancer protein polynucleotide. In some embodiments, the expression of the enhancer protein will result in a decrease in the nuclear transport of the viral polymerase. Stabilization of the system will result in the degradation of the enhancer protein, and the mRNA transport of the viral polymerase will resume. Without being bound by theory, this feedback can prevent the normal regulation of the cell while overexpressing the recombinant protein. In some situations, the use of viral polymerases offers the advantage of higher expression levels on a cell-cell basis compared to systems using eukaryotic polymerases.

[0150] Figures 4A-4D show successful expression of GFP in concert with the L protein from ECMV from the T7 promoter when co-transfected with vectors bearing T7. HEK293 cells were cultured at 0.05 x 10 in 24-well plates prior to transient transfection with either EG1 or EG3 and EG4 as described above. 6 Cells / well were seeded and incubated overnight at 37°C and 5% CO2. GFP expression was monitored after 24 and 48 hours using a fluorescent 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 concert with GFP as a target protein and the L protein of ECMV as an enhancer protein. As in the above example, the introduction of the L protein resulted in tighter regulation of expression and therefore an overall reduction in overexpression.

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

[0152] Improved membrane protein expression and membrane localization DRD1-GFP fusions were expressed in HEK293 cells. HEK293 cells were grown at 0.05×10 in 24-well plates before being transiently transfected with either EG10 or EG8 as described above. 6 Cells were seeded at 100 cells / well and incubated overnight at 37° C. and 5% CO2. DRD1-GFP expression was monitored after 24 and 48 hours using a fluorescent microscope. Images were captured using a CCD camera (Amscope) and analyzed with ISCapture (Amscope).

[0153] 5A-5D demonstrate that EG10 is unable to accurately translocate the expressed receptor. Without being bound by theory, it is believed that as a result of overexpression of the human DRD1 receptor in human cells with the EG10 construct, the cells begin to degrade or regulate the expressed target protein. Regulation of this form results in the formation of denatured proteins as inclusion bodies (FIG. 5B, red arrows). Regulation of membrane protein expression by cells in this manner can result in inactive and misfolded proteins and, consequently, unusable and poor quality expressed proteins. In contrast, co-expression of the target membrane protein with an exemplary enhancer protein resulted in accurately translocated DRD1-GFP, as seen by accurate insertion into the membrane and the absence of inclusion bodies (FIGS. 5C-5D). This example demonstrates that co-expression of an exemplary enhancer protein (L protein of ECMV) together with an exemplary target membrane protein (DRD1) resulted in improved expression and localization of the membrane protein. Without being bound by theory, the present system appears to result in tight regulation of target protein expression, thereby circumventing normal cell regulation that would result in degradation of the expressed membrane protein, making the present system suitable for high-yield expression and purification of GPCRs.

[0154] Expression of target protein and enhancer protein from different constructs To demonstrate that enhancer proteins can be encoded by separate DNA molecules, the DRD1-GFP (EG10) construct was co-expressed with the L protein from ECMV (EG11) under the control of separate promoters in separate vectors. HEK293 cells were cultured at 0.05 × 10 in 24-well plates before transient transfection with EG10 and EG11 as described above. 6 Cells / well were seeded and incubated overnight at 37° C. and 5% CO2. DRD1-GFP expression was monitored after 48 hours using a fluorescent microscope. Images were captured 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 correct membrane association. While expression of DRD1-GFP results in the formation of inclusion bodies (Figure 10A, red arrow), correct membrane association can be achieved by co-expression of L-protein. Figure 10B demonstrates that the regulatory effect of L-protein is sufficient to restore correct membrane association of DRD1 even when L-protein is expressed from a separate vector and promoter.

[0156] These results demonstrate that the methods disclosed herein can be used to express the enhancer protein and target protein disclosed herein from separate constructs to achieve improvements in the yield and / or functionality of expressed target protein.Furthermore, these results suggest that the 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 different constructs to improve the yield and / or functionality of expressed target protein.This dramatically enhances the versatility of the methods and compositions disclosed herein.

[0157] Functional activity of membrane proteins In addition to describing a correctly translocated GPCR such as DRD1, activity testing was performed using DRD1-Strep fusions. The smaller strep-tag ensures that interactions with cytosol-located G proteins are intact and functional assays can be performed. After dopamine binding, DRD1 releases the heterotrimeric G protein to its Gα subunit and its Gβγ complex. In the resting state, Gα binds GDP, but after activation, it exchanges GTP for GDP. The Gα-GTP complex interacts with adenylate cyclase (AC), leading to activation of AC activity and consequently increasing cAMP levels. Changes in intracellular cAMP levels can be measured by standard cAMP assays. Academic and industrial standards (EG5) were compared to the same target protein in co-expression with the L protein of ECMV.

[0158] DRD1-Strep fusions were 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% CO2 before being transiently transfected with either EG5 or EG6 as described above. Proteins were allowed to express for 48 hours and DRD1 activity was analyzed using the cAMP-Glo™ Assay (Promega) according to the manufacturer's instructions. After 48 hours, cells were washed with sterile PBS pH 7.2 and cells were incubated with 20 μl of 1 mM dopamine substrate solution (+dopamine; on) or PBS pH 7.2 (-dopamine; off) for 2 hours at 37° C. After incubation, cells were washed with PBS pH 7.2 followed by the addition of 20 μl lysis buffer. Lysis was performed for 15 minutes at room temperature (RT) with shaking. Then, 40 μl detection solution was added and cells were incubated for 20 minutes at RT with shaking. Prior to analysis, the reaction was stopped using 80 μl Kinase-Glo® reagent, which was incubated for 15 min at RT. Luminescence was measured using a plate reader (BioTek Synergy™ LX) and data was analyzed using standard analysis programs.

[0159] FIG. 11 demonstrates the advantage of expressing DRD1-Strep in concert with the L protein from EMCV. When dopamine is added to cells expressing DRD1, the corresponding luminescence signal falls as a result of internal cAMP release. FIG. 11 shows that co-expressing DRD1 and the L protein 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 activators, dopamine. If the assay produces a "leaky" signal, its utility for drug discovery screening is low. FIG. 11 shows that by co-expressing DRD1 and the L protein from EMCV, "leaky" activation, and therefore false negative readings, are significantly reduced when comparing a mere off signal to non-transfected cells. Thus, co-expression of an enhancer protein using the methods disclosed herein results in tighter regulation of the activation of the target DRD1 protein. Thus, the methods disclosed herein have applicability in drug discovery screening.

[0160] Example 3 Expression of DRD1-GFP using a viral promoter in combination with a viral polymerase For this example, the T7 promoter was used to express DRD1-GFP as an exemplary difficult to express target membrane protein to demonstrate that a viral polymerase such as T7 can be used to initiate transcription outside the nucleus. As in Example 1, the viral polymerase was placed under the control of a standard eukaryotic promoter and the corresponding mRNA was relied upon for nuclear export.

[0161] Figures 6A-B demonstrate successful expression of DRD1-GFP in concert with the L protein from ECMV from the T7 promoter when co-transfected with the vector bearing T7. HEK293 cells were cultured at 0.05 × 10 in 24-well plates prior to transient transfection with either EG10 or EG12 and EG4. 6 Cells were seeded at 1000 cells / well and incubated overnight at 37° C. and 5% CO2. DRD1-GFP expression was monitored after 24 and 48 hours using a fluorescent 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 concert 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 disclosed system, method and composition are compatible with a wide variety of mammalian promoters. DRD1-GFP was used as an exemplary target protein to demonstrate the compatibility of co-expression of target and enhancer proteins from different promoters. As described in Example 2, the correct expression and translocation of DRD1-GFP can be easily detected by fluorescence microscopy. The constructs used in the experiments were engineered to express DRD1 from either the CMV promoter (EG8), the EF1-α promoter (EG22) or the SV40 promoter (EG23) and to have the following elements - a nucleic acid sequence encoding DRD1-GFP, a nucleic acid sequence encoding an IRES, and a nucleic acid sequence encoding an L protein sequence. The academic standard system (EG10) was used to account for the difference between correct and imprecise membrane association.

[0163] DRD1-GFP fusions under the control of different mammalian promoters were expressed in HEK293 cells. HEK293 cells were grown in 24-well plates at 0.05×10 cells before being transiently transfected with either EG8, EG10, EG22 or EG23 as described above. 6 Cells / well were seeded and incubated overnight at 37° C. and 5% CO2. DRD1-GFP expression was monitored after 48 hours using a fluorescent microscope. Images were captured 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 the expression of an enhancer protein. Expression of DRD1-GFP from a control construct shows that DRD1 fails to localize to the outer membrane of the cell, but rather to inclusion bodies (bright green spot, Figure 12A), whereas DRD1-GFP expressed in combination with the L-protein enhancer expressed from the CMV, EF1α and SV40 (Figures 12B-D) promoters all correctly associate with the membrane, as judged by the absence of inclusion bodies. As expected, different promoters result in different expression levels and therefore the amount of DRD1-GFP (total amount of fluorescence) at the membrane varies.

[0165] Example 5 Expression of DRD1-GFP using different viral pore-blocking proteins An 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 correct expression and translocation of DRD1-GFP can be easily detected by fluorescence microscopy. An academic standard system (EG10) was used to account for the difference between correct and incorrect membrane association. HEK293 cells were transfected at 0.05×10 in 24-well plates before being transiently transfected with either EG8, EG10, EG19, EG20 or EG21 as described above. 6 Cells / well were seeded and incubated overnight at 37° C. and 5% CO2. DRD1-GFP expression was monitored after 48 hours using a fluorescent microscope. Images were captured and analyzed by an Echo Revolve microscope system.

[0166] Figure 13 demonstrates that the leader protein of ECMV (Figure 13B), Theiler's virus leader protein (Figure 13C), poliovirus 2A protease (Figure 13D) and vesicular stomatitis virus M protein (Figure 13E) are all sufficient to ensure correct membrane incorporation of DRD1-GFP, in contrast to DRD1-GFP without any of the enhancer proteins (Figure 13A).

[0167] These results show that several different viral pore blocking proteins share the ability to improve the yield, localization and / or functionality of target protein when expressed together with target protein in host cell.Without being bound by theory, it is believed that the blockage of nuclear pore resulting from the expression of any one of these enhancer proteins may bypass the normal regulation of cells that would otherwise result in the degradation of the expressed target protein.Thus, this common mechanism by which viral pore blocking proteins enhance target protein expression, localization and activity allows the methods disclosed herein to be carried out with 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. DRD1-GFP was expressed from an EG19 construct, which also encodes an enhancer protein, or from a control EG10 construct.

[0169] DRD1-GFP fusion protein was expressed in CHO-K1 cells. CHO-K1 cells were grown at 0.05 × 10 in 24-well plates before being transiently transfected with either EG10 or EG19 using Lipofectamine 3000 (Thermofisher) according to the manufacturer's instructions. 6 Cells / well were seeded and incubated overnight at 37° C. and 5% CO2. DRD1-GFP expression was monitored after 48 hours using a fluorescent microscope. Images were captured and analyzed by an Echo Revolve microscope system.

[0170] FIG. 14 demonstrates that EG10 cannot accurately translocate the expressed receptor. Interestingly, the consequences of overexpression of the human DRD1 receptor in CHO cells appear to be more severe compared to HEK cells. The EG10 construct causes the cells to start degrading or regulating the expressed target protein, resulting in the formation of denatured proteins as inclusion bodies (FIG. 14A, red arrow). Regulating the expression of membrane proteins by cells in this manner can result in inactive and misfolded proteins, and consequently, unusable and poor quality expressed proteins. In contrast, co-expression of the target membrane protein with an exemplary enhancer protein resulted in a correctly translocated DRD1-GFP, as seen by the correct insertion into the membrane and the absence of inclusion bodies (FIG. 14B). This example demonstrates that co-expression of an exemplary enhancer protein (L protein of Theiler's virus) together with an exemplary target membrane protein (DRD1) results in improved expression and localization of the membrane protein. Moreover, this example demonstrates that a variety of eukaryotic cell types (eg, HEK293 or CHO cells) can be used in the practice of the disclosed methods.

[0171] Example 7 Production of DRD1-GFP expression 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] DRD1-GFP fusions were expressed in Sf9 cells. Sf9 cells were grown at 0.4 × 10 in 6-well plates before being transiently transfected with either EG10 or EG8 using Cellfectin reagent II (Thermofisher) according to the manufacturer's instructions. 6Cells were seeded at 1000x / well and incubated for 15 min at RT. DRD1-GFP expression was monitored after 72 h using a fluorescent microscope. Images were captured and analyzed by an Echo Revolve microscope system.

[0173] FIG. 15 demonstrates that not only is EG10 unable to translocate the expressed receptor correctly, but the expressed receptor is highly toxic to the cells. The highest fluorescent signal was observed in cells that died as a result of the toxicity of the expressed gene (FIG. 15A, red arrow). In contrast, expression of DRD1-GFP using the disclosed method prevents the cytotoxicity caused by expression of DRD1-GFP, and membrane-integrated receptor is observed (FIG. 15B, red arrow). Interestingly, the consequences of overexpression of the human DRD1 receptor in Sf9 cells appear to be more severe compared to HEK cells. Unregulated expression, as in the standard system EG10, causes high cell death and consequent unusable protein. The toxic effects, as evidenced by overall cell health and membrane-bound receptor, are dramatically milder when expressing DRD1-GFP and L protein from EG8. This example demonstrates that 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, with clearly improved control of toxic effects. Moreover, this example demonstrates that the disclosed method is compatible with a variety of eukaryotic cell types.

[0174] Example 8 IL2-inducible T cell kinase (ITK) production ITK was used as an exemplary target protein to illustrate the application of the disclosed system to express soluble proteins that are typically difficult to express. ITK is a member of the TEC family of kinases and is believed to play a role in T cell proliferation and differentiation in T cells. ITK was also used to demonstrate batch-to-batch enzyme activity consistency and scalability of the methods disclosed herein. ITK was expressed in 3×10 ml, 100 ml and 1000 ml of growth medium. Furthermore, an ITK-L-his protein fusion construct (EG9) was used to demonstrate 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) for comparison.

[0175] ITK-his and ITK-L-his fusions were expressed in HEK293 cells. HEK293 cells were cultured at 2×10 in 10 ml, 100 ml or 1000 ml Expi293 medium before being transiently transfected with either EG9, EG17 or EG18 as described above. 6 Cells were seeded at 1000×g / ml and incubated overnight at 37° C., 120 rpm and 5% CO2. Cells were harvested after 48 hours (5,000×g, 15 min, 4° C.) and cell pellets were stored at −80° C. until further use.

[0176] To purify ITK, cells were resuspended in lysis buffer (40 mM Tris, 7.5; 20 mM MgCl2; 0.1 mg / ml BSA; 50 μM DTT; and 2 mM MnCl2, protease inhibitors, DNAse), lysed by sonication (2 min, 10 sec on, 10 sec off, 40% amplitude), and the crude cell extract was clarified (5,000×g, 20 min, 4° C.). 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) before loading the clarified lysate using a peristaltic pump. After loading, purification was performed on the AKTA™ system (Cytiva Life Sciences (formerly GE Healthcare)). The column was washed with 5 CV of wash buffer before elution with a continuous gradient 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 protein-containing fractions were pooled and concentrated.

[0177] Proteins were further purified by size-exclusion chromatography (SEC) (Superdex 200, ThermoFisher) using SEC buffer (40 mM Tris, 7.5; 20 mM MgCl2, 150 mM NaCl) and 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 coupled 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. The 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 reaction buffer (provided in the kit) was combined with 10 μl enzyme dilution and 10 μl ATP / PolyE4Y1 mix. The plate was incubated for 60 min at RT. 25 μl ADP-Glo ​​reagent was added and the plate was reincubated for 40 min at RT. The reaction was stopped by adding 50 μl Kinase Detection Reagent and incubating for an additional 30 min at RT. The reaction was read by luminescence with a 1 sec integration time.

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

[0179] FIG. 17A shows the final SDS-PAGE of the purification of the identified species. Note that only the P1 species is active, therefore expression of the enhancer protein in combination with ITK results in a huge increase in the expression of the active ITK species. FIG. 17B demonstrates the difference in activity by using luminescence as the primary readout. Only P1 expressed from EG17 demonstrates high activity and is therefore the only usable protein for drug screening against this kinase. Although both systems appear to express similar amounts of the protein of interest, ITK expressed using the methods disclosed herein shows greater activity than ITK protein expressed in the absence of the enhancer protein. 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. Furthermore, not only can the disclosed methods be used to produce active proteins that would otherwise be toxic, but these proteins can then be used in drug screening, such as small molecule screening, to discover novel therapeutics.

[0180] Example 9 Production of IL2-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] ITK-his fusions were expressed in CHO-K1 cells. A total of eight 150 mm plates of each construct of CHO-K1 cells were transfected with 5×10 GFP plasmid DNA prior to transient transfection with either EG17 or EG18 using Lipofectamine 3000 (Thermofisher) according to the manufacturer's instructions. 6Cells were seeded at 1000 cells / dish and incubated overnight at 37°C and 5% CO2. Cells were harvested after 48 hours by scraping, spinning down and removing the supernatant (5,000 x g, 15 min, 4C). Cell pellets were stored at -80°C until further use. To purify ITK, cells were resuspended in lysis buffer (40 mM Tris, 7.5; 20 mM MgCl2; 0.1 mg / ml BSA; 50 μM DTT; and 2 mM MnCl2, protease inhibitors, DNAse), lysed by sonication (2 min, 10 s on, 10 s off, 40% amplitude) and crude cell extract was clarified (5,000 x g, 20 min, 4°C). A 5ml His-resin column (GE Healthcare HisTrap) was equilibrated with wash buffer (40mM Tris, 7.5; 20mM MgCl2; 0.1mg / ml BSA; 50μM DTT; and 2mM MnCl2) before loading the clarified lysate using a peristaltic pump. After loading, purification was performed on an AEKTA system. The column was washed with 5CV of wash buffer before eluting with a continuous gradient 0-75% elution buffer (wash buffer + 300mM imidazole) over 20CV. Elution was completed with 5CV of 100% elution buffer.

[0182] Protein-containing fractions were analyzed by SDS-PAGE (6-12% SurePAGE, Bis-Tris, GenScript) and protein-containing fractions were pooled and concentrated. Proteins were further purified by size-exclusion chromatography (SEC) (Superdex 200, ThermoFisher) using SEC buffer (40 mM Tris, 7.5; 20 mM MgCl2, 150 mM NaCl) and fractions were analyzed by SDS-PAGE (6-12% SurePAGE, Bis-Tris, GenScript). Protein-containing fractions were pooled according to their appearance and analyzed for activity using the ITK kinase enzyme system in combination with the ADP-Glo ​​Assay™ (Promega) according to the manufacturer's instructions.

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

[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 target proteins. Without being bound by theory, ITK seems to need to form a dimer to achieve an active form. ITK is a known kinase that is toxic to cells when overexpressed. Therefore, the higher the activity of ITK, the more expression will be downregulated or made into a monomeric inactive form by the host cell.

[0185] Figure 19 demonstrates the difference in activity by using luminescence as the primary readout. Only P1 expressed from EG17 demonstrates compatible activity against the provided ΔITK positive control. Although both systems appear to express similar amounts of the protein of interest, only the presented system achieves 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.

[0186] Example 10 Production of IL2-inducible T cell kinase (ITK) in Sf9 cells Example 8 is repeated using Sf9 cells instead of HEK293. ITK-his is expressed 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 His-tagged ITK protein is performed as described in Examples 8 and 9.

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

[0188] CFTR constructs were expressed in HEK293 cells. HEK293 cells were cultured at 0.3×10 in 6-well plates before being transiently transfected with either EG25, the PCR product of the EG25 insertion, or EG24 as described above. 6Cells were seeded at 1000 cells / well and incubated overnight at 37°C and 5% CO2. CFTR expression was monitored after 24 and 48 h using a microscope. After 48 h, cells were harvested and lysed using RIPA (radioimmunoprecipitation assay) buffer (CellGene). Lysates were cleared 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-expressing L-protein and CFTR. Although the academic standard produced a broad band in Western blot, transcription and translation based on the EG25 construct resulted in a defined band, demonstrating a highly homogenous 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 difficult-to-express toxic soluble proteins was illustrated. 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 the present system can produce high yields of active NADase, an NADase-Flag fusion was cloned into the backbone of an exemplary system (EG13).

[0191] The NADase-flag construct was expressed in HEK293 cells. HEK293 cells were grown at 5×10 in a T225 flask before being transiently transfected with either EG13 as described above. 6Cells were seeded at 1000×g and incubated overnight at 37° C. and 5% CO2. NADase-flag expression was monitored after 24 and 48 hours using a microscope. Cells were harvested after 48 hours by detaching and scraping the cells using 0.5% trypsin solution for 5 minutes at 37° C. 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 inhibitors, DNAse) and lysed by sonication (2 minutes, 10 seconds on, 10 seconds off, 40% amplitude) and crude cell extracts were 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 being added to the clarified lysate. The lysate was incubated with the resin for 2 h at 4 °C with shaking. The resin was sunk and washed with 5 CV of wash buffer and the protein was eluted with 4 × 1 CV of elution buffer (wash buffer + 0.2 mg / ml 3 × Flag-peptide (Sigma)) using a spin column. The purification was analyzed by SDS-PAGE (6-12% BOLT, ThermoFisher) (Figure 8A) and protein-containing fractions were pooled. Protein concentration was measured using an A280 (NanoDrop One, FisherScientific). Protein yield was determined to be 26 mg / L expression medium. NADase activity was examined by analyzing the conversion rate of NAD+ to ADP-ribose by HPLC (Figure 8B).

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

[0193] C1-Inh-Myc-Flag fusions were expressed in HEK293 cells. HEK293 cells were cultured at 1.75×10 in 100 ml shake flasks prior to transient transfection with a vector encoding C1-Inh (OriGene; CAT#: RC203767) alone or in combination with EG11 by suspension cell transfection using methods known in the art and / or disclosed herein. 6Cells were seeded at 100 / ml and incubated overnight at 37°C, 5% CO2 and 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 (22um, nitrocellulose). To purify C1-Inh, anti-Flag resin (anti-FLAG M2 affinity gel, Millipore Sigma) was equilibrated with 20mM Tris pH7.5, 50mM NaCl before adding to the supernatant. The supernatant was incubated with the resin for 2 hours at 4°C with shaking. The resin was sunk and washed with 5CV 20mM Tris pH7.5, 50mM NaCl and the protein was eluted with 4CV 20mM Tris pH7.5, 50mM NaCl, 0.2mg / ml 3xFlag peptide. Purifications were analyzed by SDS-PAGE (SurePAGE, Bis-Tris, GenScript) and protein-containing fractions were pooled. Protein concentrations were analyzed by BCA assay (ThermoFisher) according to the manufacturer's instructions and normalized C1-Inh was tested for activity using an immunoassay (MicroVue C1-Inihibitor Plus EIA, Quidel) according to the manufacturer's instructions.

[0194] FIG. 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. FIG. 20B demonstrates the improvement of the total amount of active C1-inhibitor in the purified sample. Protein concentrations were normalized for activity assays before testing for active C1-inhibitor. The amount of active C1-inhibitor can be increased by >10% by co-expressing enhancer protein at the same time as the 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) The application of the disclosed method to express secreted proteins with correct post-translational modifications was illustrated using PSG1 as an exemplary target protein. 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 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, which is impossible to recreate when using non-human cells. In this example, the L protein from EMCV was co-expressed with PSG1 under the control of the CMV promoter.

[0196] PSG1 was expressed in HEK293 cells. HEK293 cells were grown at 1.75 × 10 in 100 ml shake flasks prior to transient transfection with a vector encoding PSG1 in concert with the L protein from EMCV. 6Cells were seeded at 100 / ml and incubated overnight at 37° C., 5% CO2 and 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). To purify PSG1, a HiTrap™ DEAE Sepharose Fast Flow IEX column (Cytiva, formerly GE Healthcare Life Sciences) was equilibrated with wash buffer (10 mM Tris pH 7.6) before loading the supernatant onto the column using a peristaltic pump. After loading, purification was performed on an AKTA™ system (Cytiva Life Sciences, formerly GE Healthcare). The column was washed with 5 CV of wash buffer before eluting with a multi-step gradient 10%, 20%, 30%, 50% and 100% elution buffer (wash buffer + 200 mM NaCl). 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 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 arrows).

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

[0199] Embodiment 1. A system for recombinant expression of a target protein in a eukaryotic cell, comprising one or more vectors, said one or more vectors comprising: a. a first polynucleotide encoding the target protein; b. a second polynucleotide encoding an enhancer protein; Including, 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; The system, wherein the first polynucleotide and the second polynucleotide are operably linked to one or more promoters.

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

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

[0202] Embodiment 4. The system of 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 of embodiment 4, wherein the NCT inhibitor is a picornavirus leader (L) protein or a functional variant thereof.

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

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

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

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

[0208] Embodiment 10. The system of 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 of embodiment 4, wherein the NCT inhibitor is herpes simplex virus (HSV) ICP27 protein or a functional variant thereof.

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

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

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

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

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

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

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

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

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

[0219] Embodiment 21. The system of embodiment 20, wherein the expression cassette comprises a coding polynucleotide comprising the first polynucleotide and the second polynucleotide linked by a polynucleotide encoding a ribosomal skipping site, the coding polynucleotide being operably linked to the shared promoter.

[0220] Embodiment 22. The system of embodiment 20, wherein the expression cassette comprises a coding polynucleotide, the coding polynucleotide encoding the enhancer protein and the target protein linked by a ribosomal skipping site, the coding polynucleotide being operably linked to the shared promoter.

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

[0222] Embodiment 24. A system described in any one of embodiments 1 to 23, comprising one type of vector.

[0223] 25. A method for the production of a vector comprising: a. a first vector comprising said first polynucleotide operably linked to a first promoter; and 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, comprising:

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

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

[0226] Embodiment 28. The system of any one of embodiments 1 to 27, wherein at least one of the one or more vectors comprises 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 of any one of embodiments 1 to 28, wherein at least one of the one or more vectors comprises 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; A vector, wherein the first polynucleotide and the second polynucleotide are operably linked to at least one promoter.

[0229] Embodiment 31. The vector of embodiment 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.

[0230] Embodiment 32. The vector of 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 expressing 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, wherein the exogenous polynucleotide is operably linked to a promoter.

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

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

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

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

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

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

[0238] Embodiment 40. A eukaryotic cell produced by introducing the system described in any one of embodiments 1 to 29 or the vector described in any one of embodiments 30 to 32.

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

[0240] Embodiment 42. A method for expressing a target protein in a eukaryotic cell, comprising introducing into the eukaryotic cell a polynucleotide encoding the target protein, the polynucleotide being operably linked to a promoter; the method utilizing 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 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 of embodiment 42, wherein said co-expression of an enhancer protein comprises introducing into said eukaryotic cell a polynucleotide encoding said enhancer protein operably linked to a promoter.

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

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

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

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

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

[0247] Embodiment 49. The system of any one of embodiments 1 to 29, the vector of any one of embodiments 30 to 32, the eukaryotic cell of any one of embodiments 33 to 35, the method of any one of embodiments 36 to 39 and 42 to 44, the eukaryotic cell of embodiment 40, and the target protein of 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 to the amino acid sequence of SEQ ID NO: 18.

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

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

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

[0251] Embodiment 53. A method for generating antibodies against a target protein, comprising the step of immunizing a subject with a cell described in any one of embodiments 33 to 35, a cell described in embodiment 40, or a target protein described in embodiment 41.

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

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

[0254] Embodiment 56. The method of any one of embodiments 53-55, comprising 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. A cell according to any one of embodiments 33 to 35, a eukaryotic cell according to embodiment 40, or a target protein according to embodiment 41, wherein the cell or the target protein is labeled; b. a population of recombinant cells, wherein the recombinant cells express a library of polypeptides, each of which comprises an antibody or antigen-binding fragment thereof; providing a solution comprising: isolating one or more recombinant cells from the solution by sorting for the labeled cells or recombinant cells bound to the labeled target protein. The method includes:

[0256] Embodiment 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 embodiments 33 to 35, with a eukaryotic cell according to embodiment 40, or with a target protein according to embodiment 41; b. purifying and / or enriching members of the phage display library that bind to the cell or target protein; The method includes:

[0257] Embodiment 59. The eukaryotic cell of 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 of any one of embodiments 33 to 35, 40 and 59, which is a eukaryotic cell line.

[0259] Embodiment 61. A eukaryotic cell according to any one of embodiments 33 to 35, 40, 59 and 60, which is a 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 of embodiment 60, wherein the eukaryotic cell line is a stable cell line.

[0261] Embodiment 63. The system of 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 herpes virus vector, a baculovirus vector or a non-viral plasmid.

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

[0263] Embodiment 65. The vector described in any one of embodiments 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 herpes virus vector, a baculovirus vector or a non-viral plasmid.

[0264] Embodiment 66 The vector described in embodiment 65, which is an AAV vector.

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

[0266] Embodiment 68. The system of 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, said one or more vectors comprising: a. a first polynucleotide encoding the target protein; b. a second polynucleotide encoding an L protein of an encephalomyocarditis virus (EMCV), optionally wherein the L protein shares at least 90% identity with SEQ ID NO:2; and wherein 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, said one or more vectors comprising: a. a first polynucleotide encoding the target protein; b. a second polynucleotide encoding the L protein of Theiler's virus, optionally wherein the L protein shares at least 90% identity with SEQ ID NO:1; and wherein the first polynucleotide and the second polynucleotide are operably linked to one or more promoters.

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

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

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

[0272] Embodiment 74. The system of any one of embodiments 69-72, 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 to the amino acid sequence of SEQ ID NO:18.

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

[0274] Embodiment 76. A system described in any one of embodiments 69 to 72, wherein the target protein is ITK, and optionally, the ITK comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 15.

[0275] Embodiment 77. A system described in any one of embodiments 69 to 72, wherein the target protein is an NADase, and optionally the NADase comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 20.

Claims

1. A system for recombinant expression of a target protein in a eukaryotic cell, comprising: a) a first polynucleotide encoding the target protein; b) a second polynucleotide encoding a nucleocytoplasmic transport (NCT) inhibitor protein, said NCT inhibitor protein being a picornavirus leader (L) protein or a functional variant thereof having at least 90% sequence identity to the amino acid sequence of said picornavirus L protein, wherein co-expression of said NCT inhibitor protein with said target protein enhances the yield and / or functionality of said target protein; and wherein the first polynucleotide and the second polynucleotide are operably linked to one or more promoters.

2. A system for recombinant expression of a target protein in a eukaryotic cell comprising one or more vectors, said one or more vectors comprising: a) a first polynucleotide encoding the target protein; b) a second polynucleotide encoding a nucleocytoplasmic transport (NCT) inhibitor protein, said NCT inhibitor protein being a picornavirus leader (L) protein or a functional variant thereof having at least 90% sequence identity to the amino acid sequence of said picornavirus L protein, wherein co-expression of said NCT inhibitor protein with said target protein enhances the yield and / or functionality of said target protein; and wherein the first polynucleotide and the second polynucleotide are operably linked to one or more promoters.

3. The system described in claim 1 or 2, wherein the picornavirus L protein is an L protein of encephalomyocarditis virus (EMCV) and / or the picornavirus L protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

2.

4. The system described in claim 1 or 2, wherein the picornavirus L protein is a Theiler's virus L protein and / or the picornavirus L protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

1.

5. The system according to any one of claims 1 to 4, wherein the system comprises a vector comprising an expression cassette, the expression cassette comprising the first polynucleotide and the second polynucleotide.

6. the expression cassette comprises a first promoter operably linked to the first polynucleotide and a second promoter operably linked to the second polynucleotide; or the expression cassette comprises a promoter operably linked to both the first polynucleotide and the second polynucleotide; The system of claim 5.

7. 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; and The system according to any one of claims 1 to 4, comprising:

8. The system according to any one of claims 1 to 7, wherein the first polynucleotide and / or the second polynucleotide is operably linked to an internal ribosome entry site (IRES).

9. 9. The system of any one of claims 2 to 8, wherein the one or more vectors comprise a T7 promoter configured for transcription of the first polynucleotide and / or the second polynucleotide by T7 RNA polymerase.

10. The system according to any one of claims 2 to 9, wherein the one or more vectors comprise a polynucleotide sequence encoding T7 RNA polymerase.

11. 11. The system of any one of claims 2 to 10, 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 herpes virus vector, a baculovirus vector, or a non-viral plasmid.

12. The system according to any one of claims 1 to 11, wherein the target protein is a secreted protein, an intracellular protein, an enzyme or a membrane protein.

13. A eukaryotic cell for the expression of a target protein comprising a system according to any one of claims 1 to 12.

14. A eukaryotic cell for expressing a target protein, comprising an exogenous polynucleotide encoding a nucleocytoplasmic transport (NCT) inhibitor protein, wherein the NCT inhibitor protein is a picornavirus leader (L) protein or a functional variant thereof having at least 90% sequence identity to the amino acid sequence of the picornavirus L protein, wherein co-expression of the NCT inhibitor protein and the target protein enhances the yield and / or functionality of the target protein, and wherein the exogenous polynucleotide is operably linked to a promoter.

15. A method for the recombinant expression of a target protein, comprising the step of introducing a system according to any one of claims 1 to 12 into a eukaryotic cell in vitro or ex vivo.

16. A method for recombinant expression of a nucleocytoplasmic transport (NCT) inhibitor protein, comprising introducing into a eukaryotic cell in vitro or ex vivo a polynucleotide encoding said NCT inhibitor protein operably linked to a promoter, said NCT inhibitor protein being a picornavirus leader (L) protein or a functional variant thereof having at least 90% sequence identity to the amino acid sequence of said picornavirus L protein, wherein co-expression of said NCT inhibitor protein with said target protein enhances the yield and / or functionality of said target protein. method.

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