Method for optimizing antibody expression

Abstract

Disclosed herein are methods, compositions, and components for optimizing or increasing expression of proteins, polypeptides, or fragments from the proteins, polypeptides.

Description

[0001] Citation of Related Applications

[0002] This application is a divisional application of the Chinese invention patent application with application number 201980041409.9, application date May 1, 2019, and invention name “A method for optimizing antibody expression”, and the original application is a Chinese national phase application with international application number PCT / US2019 / 030295. This application claims the benefit of U.S. Provisional Application No. 62 / 665,093, entitled “A Method for Optimizing Antibody Expression”, filed on May 1, 2018, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy was created on April 30, 2019, is named _AMBX_0226_00PCT_ST25.txt and is 15,184 bytes in size. Field of the Invention

[0005] The present disclosure relates to the field of molecular biology. The present disclosure relates to the design and engineering of vectors and plasmids. The present disclosure also relates to novel production strains or cells incorporating non-naturally encoded amino acids, and methods for producing recombinant proteins from the production strains or cells. Background of the Invention

[0007] The accumulation of complete, correctly assembled active antibody fragments in the periplasm of E. coli requires multiple steps to occur correctly, including: transcription, translation, stop codons that inhibit the incorporation of unnatural amino acids, trans-inner membrane transport, cleavage of signal peptides, correct folding, correct cis or trans isomerization of proline residues, assembly of light and heavy chains, correct formation of intra- and inter-disulfide bonds, and retention in the periplasm (no trans-membrane penetration into the culture medium). Alternative pathways for polypeptides in the cytoplasm or periplasm include truncation, misfolding, aggregation, and proteolysis (Miot M. and Betton JM, Microbial Cell Factories, 3, 4, 2004; Yoon, SH et al., Recent Patents in Biotech, 4, 23, 2010).

[0008] Many kinds of auxiliary proteins and / or factors in Escherichia coli are used to help produce proteins in their correct folding and active state. These proteins include chaperones, folding enzymes, isomerases, etc., but are not limited thereto. Chaperones bind to misfolded or unfolded proteins and help to fold correctly. Other auxiliary proteins and / or factors include proteins involved in disulfide bond reduction / oxidation or isomerization, peptidyl-prolyl isomerases that interconvert cis / trans peptidyl-prolyl bonds, and proteases that degrade misfolded or aggregated proteins. Overexpression of many of these proteins has been used to increase the titer of heterologous proteins (including antibody fragments) in Escherichia coli (Thomas et al., Applied Biochemistry and Biotechnology, 66, 197, 1997; Kolaj et al., Microbial Cell Factories, 8, 2009; Merdanovic et al., Annu. Rev. Microbiol. 65, 149, 2011).

[0009] In industry, prokaryotic expression of antibodies or antibody fragments incorporating unnatural amino acids at selected or specific sites is difficult to achieve. This is especially true for antibody fragments in E. coli. To date, only a few biotechnology and pharmaceutical companies have been able to successfully express wild-type antibody fragments in E. coli with high titers.

[0010] To overcome the challenges of expressing difficult-to-express (DTE) proteins, the present invention utilizes accessory proteins and / or factors (including chaperone proteins) in the design and engineering of vectors and plasmids generated by the vectors to achieve optimization of the potency of antibody fragments into which non-naturally encoded amino acids are incorporated. Summary of the invention

[0011] The present invention provides novel engineered vectors, plasmids and production strains. In some embodiments, novel expression plasmids of the present invention and corresponding production strains are used for protein expression optimization and titer yield. In embodiments of the present invention, the engineered vectors and strains comprise a nucleic acid sequence encoding a target protein and a nucleic acid sequence encoding one or more components, wherein the target protein has a non-natural amino acid site specifically incorporated, wherein the component is an auxiliary protein or factor or genetic element. In other embodiments, the present disclosure provides methods and expression plasmids and production strains for optimizing protein expression in prokaryotic systems (such as but not limited to Escherichia coli).

[0012] In some embodiments, the one or more components are eukaryotic or prokaryotic components. In some embodiments, the one or more components are auxiliary proteins or factors or genetic elements. The genetic elements can be genes or polynucleotides encoding proteins or regulatory elements.

[0013] In one embodiment, the present invention provides a vector for optimizing protein expression, the vector comprising a nucleic acid sequence encoding a target protein and a nucleic acid sequence encoding one or more components, the target protein having a specifically incorporated non-naturally encoded amino acid site, wherein the one or more components are selected from SEQ.ID.NO.: 12, 15 or 17.

[0014] In other embodiments, the one or more components may be involved in protein folding, periplasmic chaperones, genetic elements comprising DNA coding sequences, Fab heavy chain (HC) carboxyl terminal extensions, and plasmid partitioning locus parB. In some embodiments, one of these components may be included in an expression plasmid along with the protein of interest. In other embodiments, two or three or more of these components may be included in a plasmid along with the protein of interest. In some embodiments, all components may be included in a single expression plasmid with a native promoter or in a two plasmid system with an inducible promoter.

[0015] In other embodiments, the one or more components are auxiliary proteins or factors or genetic elements. In other embodiments, the auxiliary proteins or factors are chaperones. The chaperones are Skp or FkpA or Skp and FkpA. In another embodiment, the genetic element is the distribution B locus (parB).

[0016] In one embodiment, the vector of the present invention further comprises a heavy chain C-terminal extension variant. In another embodiment, the heavy chain C-terminal extension variant is an antibody heavy chain C-terminal extension variant. In other embodiments, the heavy chain C-terminal extension variant comprises one or more amino acids.

[0017] In other embodiments, parB is located between the Afe1 site and the Not1 site of the nucleic acid sequence encoding the target protein in a forward transcription orientation. In another embodiment, parB is located at the Afe1 site or the Zra1 site of the nucleic acid sequence encoding the target protein in a forward transcription orientation. In another embodiment, parB is located at the Afe1 or Zra1 site of the nucleic acid sequence encoding the target protein in a reverse transcription orientation. In another embodiment, the vector further comprises a partitioning B locus (parB).

[0018] In one embodiment, one or more proteins capable of promoting protein folding are FkpA, Skp, SurA, PPiA and PPiD. In other embodiments, the one or more proteins capable of promoting protein secretion or translocation are SecY, SecE, SecG, SecYEG, SecA, SecB, FtsY and Lep. In another embodiment, the one or more proteins capable of promoting disulfide bond formation are DsbA, DsbB, DsbD, DsbG. In some embodiments, the chaperone protein may include Skp or FpkA. In other embodiments, parB can be cloned in the forward direction between the Afe1 site and the Not1 site, at the Zra1 site or at the Afe1 site. In other embodiments, the plasmid contains a sequence deletion of 271 base pairs. In another embodiment, the plasmid contains parB in the forward direction and Skp or FpkA chaperone proteins cloned at the Zra1 site. In other embodiments, the plasmid contains parB or FkpA in an upstream or downstream position and parB in a forward orientation. In another embodiment, the plasmid contains FkpA in an upstream or downstream position and in a forward or reverse orientation. In another embodiment, the plasmid contains parB in an upstream position and FkpA in a downstream position. In another embodiment, the plasmid contains parB in a downstream position and FkpA in an upstream position. In some embodiments, the heavy chain carboxyl terminal extension may comprise or consist of 1, 2, 3, 4, 5 or more amino acids. In some embodiments, the heavy chain carboxyl terminal extension may comprise or consist of 5 or more amino acids having at least one mutation therein.

[0019] In other embodiments, the target protein is selected from a biotherapeutic agent, a bioactive molecule, an immunogen, an antibody, an antibody fragment or a variant thereof. In other embodiments, the biotherapeutic agent is a vaccine. In another embodiment, the target protein is a cytokine, a chemokine, a growth factor, a growth factor receptor, an interferon, an interleukin, an inflammatory molecule, an oncogene product, a peptide hormone, a signal transduction molecule or a steroid hormone receptor. In other embodiments, the target protein is HER2, CD-70, PSMA, 5T4, EGFR, TROP2, CD3, IL-2, IL-3, IL-10, IL-12, IL-15, IL-21, GPC3, DLL3, ROR1, leptin, growth hormone releasing peptide, FGF-1, FGF-19, FGF-21, FGF-23, HGH, FcR, insulin, IGF1, TNFR1, TRAIL, EPO and their analogs, bispecific molecules or fragments.

[0020] In one embodiment, the antibody is a full-length antibody or an antibody fragment. In other embodiments, the antibody is an IgG1, IgG2, IgG3 or IgG4 antibody. In other embodiments, the antibody fragment is Fab, Fab', F(ab')2, Fv fragment, single-chain antibody fragment (scFv), disulfide-stabilized scFv (dsFv)), double antibody (Db), BiTE (bispecific T cell adapter), DART (dual affinity retargeting) or tandem double antibody (TandAb). In some embodiments, the antibody fragment is Fab.

[0021] In embodiments of the invention, the engineered plasmids and strains comprise a nucleic acid sequence encoding an antibody targeting a target protein or polypeptide and a nucleic acid sequence encoding one or more components, wherein the target protein or polypeptide has a non-natural amino acid site specifically incorporated, and the target protein or polypeptide includes but is not limited to HER2, CD-70, PSMA, 5T4, EGFR, TROP2, CD3, IL-2, IL-3, IL-10, IL-12, IL-15, IL-21, GPC3, DLL3, ROR1, leptin, growth hormone releasing peptide, FGF-1, FGF-19, FGF-21, FGF-23, HGH, FcR, insulin, IGF1, TNFR1, TRAIL, EPO and their analogs, bispecific molecules or fragments, wherein the one or more components are auxiliary proteins or factors or genetic elements. In other embodiments of the invention, the engineered plasmid comprises a nucleic acid sequence encoding an antibody specific for any target antigen having a non-natural amino acid site specifically incorporated therein, including but not limited to HER2, CD-70, PSMA, 5T4, EGFR, TROP2, CD3, IL-2, IL-3, IL-10, IL-12, IL-15, IL-21, GPC3, DLL3, ROR1, leptin, growth hormone releasing peptide, FGF-1, FGF-19, F GF-21, FGF-23, HGH, FcR, insulin, IGF1, TNFR1, TRAIL, EPO and their analogs, bispecific molecules or fragments, wherein the fragment is Fab, Fab', F(ab')2, Fv fragment, single-chain antibody fragment (scFv), disulfide-stabilized scFv (dsFv)), diabody (Db), BiTE (bispecific T cell engager), DART (dual affinity retargeting) or tandem diabody (TandAb), etc., wherein one or more components are auxiliary proteins or factors or genetic elements. In other embodiments, the nucleic acid sequence encodes HER2, CD-70, PSMA, 5T4, EGFR, TROP2, CD3, IL-2, IL-3, IL-10, IL-12, IL-15, IL-21, GPC3, DLL3, ROR1, leptin, growth hormone releasing peptide, FGF-1, FGF-19, FGF-21, FGF-23, HGH, FcR, insulin, IGF1, TNFR1, TRAIL, EPO and their analogs, bispecific molecules or antibody fragments, but are not limited thereto.In other embodiments, the antibody fragment is Fab, Fab', F(ab')2, Fv fragment, single-chain antibody fragment (scFv), disulfide-stabilized scFv (dsFv)), diabody (Db), BiTE (bispecific T cell engager), DART (dual affinity retargeting) or tandem diabody (TandAb), but is not limited thereto. In other embodiments, the antibody or fragment thereof is a PEGylated Fab molecule. In other embodiments, the antibody fragment is an anti-CD3 Fab.

[0022] In one embodiment, the engineered production strain or cell is 2797, 2835, 2884, 2885, 2886, 2887, 2909, 2910, 2914, 2915, 2916, 2917, 2918, 2839, 2840, 2841, 3004, 3005 or 3006 as described in the figures, tables and examples herein. In another embodiment, the expression plasmid is plasmid p56, p96, p117, p118, p119, p120, p134, p135, p141, p142, p143, p144, p145, p94, p199, p200 or p201 as described in the figures, tables and examples herein.

[0023] In one embodiment, the present invention provides a recombinant cell comprising a nucleic acid sequence encoding a target protein and a nucleic acid sequence encoding one or more components, wherein the target protein has a non-naturally encoded amino acid site specifically incorporated, wherein the one or more components are selected from SEQ.ID.NO.: 12, 15 or 17. In one embodiment, the recombinant cell is strain 2797, 2835, 2884, 2885, 2886, 2887, 2909, 2910, 2914, 2915, 2916, 2917, 2918, 2839, 2840, 2841, 3004, 3005 or 3006. In other embodiments, the recombinant cell comprises a chaperone protein Skp and a partitioning B locus (parB). The cell is selected from strain 2910 or 2841. In other embodiments, the recombinant cell comprises a chaperone protein FkpA and a partitioning B locus (parB). The cell is selected from strains 2909, 2840, 3004, 3005 or 3006. In other embodiments, the recombinant cell comprises a heavy chain C-terminal extension variant. The cell is selected from strains 2914, 2915, 2916, 2917 or 2918.

[0024] In one embodiment, the invention provides an expression plasmid according to any one of the claims, wherein the plasmid is p56, p94, p96, p117, p118, p119, p120, p134, p135, p141, p142, p143, p144, p145, p199, p200 or p201.

[0025] In other embodiments, optimizing or increasing the expression titer of a target protein, polypeptide, or antibody fragment in a prokaryotic system such as E. coli can include optimizing or increasing the expression yield by at least 0.2 times, at least 0.5 times, at least 1 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or more, relative to the starting strain. In other embodiments, optimizing or increasing the expression titer of a target protein, polypeptide, or antibody fragment in a prokaryotic system such as E. coli can include optimizing or increasing the expression yield by about or approximately 0.25 g / L, 0.5 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L, 3.0 g / L, 3.5 g / L, 4.0 g / L, 4.5 g / L, 5.0 g / L or greater, 10 g / L or greater, 20 g / L or greater.

[0026] In one embodiment, the present invention provides a method for producing a target recombinant protein incorporating non-naturally encoded amino acids, the method comprising expressing the protein in a bacterial cell and introducing a nucleic acid sequence encoding one or more components, wherein the one or more components are selected from SEQ.ID.NO.: 12, 15 or 17. In another embodiment, a method for increasing the yield of a protein with a non-natural amino acid site specifically incorporated is provided. In some embodiments, the present invention provides a method for optimizing or increasing the expression of a recombinant protein of a target protein that is difficult to express, wherein the protein incorporates non-naturally encoded amino acids. In other embodiments, the position and orientation of the chaperone gene relative to the target gene can optimize the yield of the recombinant protein.

[0027] In other embodiments, the target protein, polypeptide, antibody or gene is a cytokine, a growth factor, a growth factor receptor, an interferon, an interleukin, an inflammatory molecule, an oncogene product, a peptide hormone, a signal transduction molecule, a steroid hormone receptor, a transcription activator, a transcription repressor, erythropoietin (EPO), insulin, human growth hormone, epithelial neutrophil activation peptide-78, GROα / MGSA, GROβ, GRO, MIP-1α, MIP-1, MCP-1, hepatocyte growth factor, insulin-like growth factor, leukemia inhibitory factor, oncostatin M, PD-ECSF, PDGF, pleiotropin, SCF, c-kit ligand, VE GF, G-CSF, IL-1, IL-2, IL-8, IGF-I, IGF-II, FGF (fibroblast growth factor), TNF, TGF-α, TGF-β, EGF (epidermal growth factor), KGF (keratinocyte growth factor), SCF / c-Kit, CD40L / CD40, VLA-4 / VCAM-1, ICAM-1 / LFA-1, hyaluronic acid / CD44, Mos, Ras, Raf, Met, p53, Tat, Fos, Myc, Jun, Myb, Rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and corticosterone, alpha-1 antitrypsin, angiostatin, antihemolytic Factor, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic peptide, atrial peptide, CXC chemokine, T39765, NAP-2, ENA-78, Gro-a or Gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1α, monocyte inflammatory protein-1β, RANTES, I309, R83915, R91733, HCC1, T58847, D31065, T64262, C-kit ligand, collagen, Colony stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activation peptide-78, epidermal growth factor (EGF), epithelial neutrophil activation peptide, erythropoietin (EPO), shedding toxin, factor IX, factor VII, factor VIII, factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, G-CSF, GM-CSF, glucocerebrosidase, gonadotropin, growth factor, growth factor receptor, Hedgehog protein, hemoglobin, hepatocyte growth factor (HGF), hirudin, human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 receptor,Insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon, IFN-α, IFN-β, IFN-γ, interleukin, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurotrophin, neutrophil inhibitory factor (NIF), oncostatin M, osteogenic protein, oncogene products, parathyroid hormone, PD-ECSF, PDGF, peptide hormones, human growth hormone, pleiotrophin, protein A, protein G, pyrogenic exotoxin A, B or C, relaxin, renin, SCF, soluble complement receptor I, soluble I-CAM 1. Soluble interleukin receptor, soluble TNF receptor, somatomedin, somatostatin, growth hormone, streptokinase, superantigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin α1, tissue plasminogen activator, tumor growth factor (TGF), TGF-α, TGF-β, tumor necrosis factor, tumor necrosis factor α, tumor necrosis factor β, tumor necrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular endothelial growth factor (VEGF), urokinase, Mos, Ras, Raf, Met, p53, Tat, Fos, Myc, Jun, Myb, Rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and corticosterone, etc.

[0028] In other embodiments, the unnatural amino acid is selected from the group consisting of: O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-DOPA (L-Dopa), fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L -phosphoserine, phosphonoserine, phosphonotyrosine, p-iodophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, unnatural analogs of tyrosine amino acids; unnatural analogs of glutamine amino acids; unnatural analogs of phenylalanine amino acids; unnatural analogs of serine amino acids; unnatural analogs of threonine amino acids; alkyl, aryl, acyl, azido, cyano, halide, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, Thioacid, borate, boronate, phosphorus, phosphono, phosphine, heterocycle, enone, imine, aldehyde, hydroxylamine, keto or amino substituted amino acids or any combination thereof; amino acids with photoactivatable crosslinkers; spin-labeled amino acids; fluorescent amino acids; amino acids with novel functional groups; amino acids that covalently or non-covalently interact with another molecule; metal binding amino acids; metal-containing amino acids; radioactive amino acids; photocaged amino acids and / or photoisomerizable amino acids; amino acids containing biotin or biotin analogs; glycosylated or carbohydrate-modified amino acids; keto-containing amino acids; amino acids containing polyethylene glycol or polyethers; heavy atom substituted amino acids; chemically cleavable or photocleavable amino acids; amino acids with extended side chains; amino acids containing toxic groups; sugar-substituted amino acids, such as sugar-substituted serine, etc.; carbon-linked sugar-containing amino acids; redox-active amino acids; α-hydroxyl-containing acids; amino thioacid-containing amino acids; α,α-disubstituted amino acids; β-amino acids; and cyclic amino acids other than proline.

[0029] In other embodiments, the invention provides a cell or cell line for separation. In some embodiments, the cell or cell line for separation is used to produce a protein or polypeptide containing a non-natural amino acid. In another embodiment, the invention provides a cell or cell line for separation, wherein the yield of a protein containing a non-natural amino acid is at least 0.25 times or higher in the presence of one or more chaperone proteins and a parB locus. In other embodiments, the yield of a protein containing a non-natural amino acid is at least 0.25 times or higher in the presence of one or more chaperone proteins, and the parB locus is located at the Afe1 site of a nucleic acid sequence encoding a target protein with an inverted orientation. In other embodiments. The cell or cell line for separation optimizes the retention of plasmids in the production cell or cell line.

[0030] This application also includes the following implementation methods:

[0031] 1. A vector for optimizing protein expression, the vector comprising a nucleic acid sequence encoding a target protein and a nucleic acid sequence encoding one or more components, the target protein having a non-naturally encoded amino acid site specifically incorporated, wherein the one or more components are selected from SEQ.ID.NO.: 12, 15 or 17.

[0032] 2. A vector as described in embodiment 1, wherein the one or more components are auxiliary proteins or factors or genetic elements.

[0033] 3. A vector as described in embodiment 2, wherein the auxiliary protein or factor is a chaperone protein.

[0034] 4. The vector of embodiment 2, wherein the genetic element is the partitioning B locus (parB).

[0035] 5. The vector of embodiment 1 further comprises an antibody heavy chain C-terminal extension variant.

[0036] 6. A vector as described in embodiment 5, wherein the heavy chain C-terminal extension variant comprises one or more amino acids.

[0037] 7. The vector as described in embodiment 1, wherein parB is located between the Afe1 and Not1 sites of the nucleic acid sequence encoding the target protein in a forward transcription orientation.

[0038] 8. The vector as described in embodiment 1, wherein parB is located at the Afe1 or Zra1 site of the nucleic acid sequence encoding the target protein in a forward transcription orientation.

[0039] 9. The vector as described in embodiment 1, wherein parB is located at the Afe1 or Zra1 site of the nucleic acid sequence encoding the target protein in a reverse orientation.

[0040] 10. The vector of embodiment 3, wherein the chaperone protein is Skp or FkpA.

[0041] 11. The vector of embodiment 3, wherein the chaperone proteins are Skp and FkpA.

[0042] 12. The vector of embodiment 10 or 11, further comprising a partitioned B locus (parB).

[0043] 13. The vector of embodiment 1, wherein the target protein is selected from a biotherapeutic agent, an immunogen, an antibody, an antibody fragment, or a variant thereof.

[0044] 14. The vector of embodiment 13, wherein the biotherapeutic agent is a vaccine.

[0045] 15. The vector of embodiment 13, wherein the target protein is a cytokine, a chemokine, a growth factor, a growth factor receptor, an interferon, an interleukin, an inflammatory molecule, an oncogene product, a peptide hormone, a signal transduction molecule or a steroid hormone receptor.

[0046] 16. The vector of embodiment 13, wherein the target protein is HER2, CD-70, PSMA, 5T4, EGFR, TROP2, CD3, IL-2, IL-3, IL-10, IL-12, IL-15, IL-21, GPC3, DLL3, ROR1, leptin, growth hormone releasing peptide, FGF-1, FGF-19, FGF-21, FGF-23, HGH, FcR, insulin, IGF1, TNFR1, TRAIL, EPO and their analogs, bispecific molecules or fragments.

[0047] 17. A vector as described in embodiment 13, wherein the antibody fragment is Fab, Fab', F(ab')2, Fv fragment, single-chain antibody fragment (scFv), disulfide-stabilized scFv (dsFv)), diabody (Db), BiTE (bispecific T cell engager), DART (dual affinity retargeting) or tandem diabody (TandAb).

[0048] 18. A vector as described in embodiment 17, wherein the antibody fragment is Fab.

[0049] 19. A vector as described in embodiment 13, wherein the antibody fragment is anti-CD3Fab.

[0050] 20. A recombinant cell comprising a nucleic acid sequence encoding a target protein and a nucleic acid sequence encoding one or more components, wherein the target protein has a non-naturally encoded amino acid site specifically incorporated, wherein the one or more components are selected from SEQ.ID.NO.: 12, 15 or 17.

[0051] 21. The recombinant cell of embodiment 20, wherein the cell is strain 2797, 2835, 2884, 2885, 2886, 2887, 2909, 2910, 2914, 2915, 2916, 2917, 2918, 2839, 2840, 2841, 3004, 3005, or 3006.

[0052] 22. The recombinant cell of embodiment 20, wherein the cell comprises the chaperone Skp and the partitioning B locus (parB).

[0053] 23. The recombinant cell of embodiment 22, wherein the cell is selected from strain 2910 or 2841.

[0054] 24. A recombinant cell as described in embodiment 20, wherein the cell comprises the chaperone protein FkpA and the partitioning B locus (parB).

[0055] 25. The recombinant cell of embodiment 24, wherein the cell is selected from strains 2909, 2840, 3004, 3005 or 3006.

[0056] 26. A recombinant cell as described in embodiment 20, wherein the cell comprises an antibody heavy chain C-terminal extension variant.

[0057] 27. The recombinant cell of embodiment 26, wherein the cell is selected from strain 2914, 2915, 2916, 2917 or 2918.

[0058] 28. An expression plasmid according to any one of the preceding embodiments, wherein the plasmid is p56, p94, p96, p117, p118, p119, p120, p134, p135, p141, p142, p143, p144, p145, p199, p200 or p201.

[0059] 29. A method for producing a target recombinant protein incorporating non-naturally encoded amino acids, the method comprising expressing the protein in a bacterial cell and introducing a nucleic acid sequence encoding one or more components, wherein the one or more components are selected from SEQ.ID.NO.: 12, 15 or 17.

[0060] 30. The method of embodiment 29, which is used to increase the yield of a protein having a specifically incorporated non-natural amino acid site.

[0061] 31. A method as described in embodiment 30, wherein the non-natural amino acid is O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, p-propargyloxy-L-phenylalanine, tri-O-acetyl-GlcNAcβ-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphotyrosine , p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, p-acetylphenylalanine, p-nitrophenylalanine, p-sulfotyrosine, p-carboxyphenylalanine, o-nitrophenylalanine, m-nitrophenylalanine, p-borylphenylalanine, o-borylphenylalanine, m-borylphenylalanine, p-aminophenylalanine, o-aminophenylalanine, m-aminophenylalanine, p-acylphenylalanine, o-acylphenylalanine, m-acylphenylalanine, p-OMephenylalanine, o-OMephenylalanine, m-OMephenylalanine, p-sulfophenylalanine, o-sulfophenylalanine phenylalanine, m-sulfophenylalanine, 5-nitroHis, 3-nitroTyr, 2-nitroTyr, nitro-substituted Leu, nitro-substituted His, nitro-substituted De, nitro-substituted Trp, 2-nitroTrp, 4-nitroTrp, 5-nitroTrp, 6-nitroTrp, 7-nitroTrp, 3-aminotyrosine, 2-aminotyrosine, o-sulfotyrosine, 2-sulfophenylalanine, 3-sulfophenylalanine, o-carboxyphenylalanine, m-carboxyphenylalanine, p-acetyl-L-phenylalanine, p-propargyl-phenylalanine, o-methyl-L-tyrosine phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine or p-propargyloxy-phenylalanine.

[0062] phenylalanine, p- ...

[0063] Acid, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine or p-propargyloxy-phenylalanine.

[0064] 32. The method of embodiment 31, wherein the non-natural amino acid is p-acetylphenylalanine.

[0065] 33. An isolated cell or cell line according to claim 29.

[0066] 34. The isolated cell or cell line of embodiment 33, wherein the isolated cell or cell line is used to produce a protein or polypeptide containing a non-natural amino acid.

[0067] 35. The isolated cell or cell line of embodiment 29, wherein the production of the protein containing the unnatural amino acid is at least 0.25 times or more in the presence of one or more chaperone proteins and the parB locus.

[0068] 36. The isolated cell or cell line of embodiment 35, wherein the parB locus is located in a reverse orientation at the Afe1 site of the nucleic acid sequence encoding the target protein.

[0069] 37. The isolated cell or cell line of embodiment 35, which is used to optimize plasmid retention in the production cell or cell line.

[0070] 38. A vector as described in embodiment 17, wherein the antibody is a full-length antibody.

[0071] 39. The vector of embodiment 38, wherein the full-length antibody is an IgG1, IgG2, IgG3 or IgG4 antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description of illustrative embodiments that utilize the principles of the present disclosure and the accompanying drawings provided:

[0073] Figure 1 Schematic representation of the anti-CD3 Fab-HK129pAcF expression cassette, wherein the unnatural amino acid pAcF incorporation site is shown within the heavy chain constant domain (CH1) region.

[0074] Figure 2 . General protocol for the construction of expression plasmids and their corresponding production strains.

[0075] Figure 3 .Circular diagram of plasmid p56 showing the names, locations and transcription directions of the various expression elements.

[0076] Figure 4 Fermentation titers of production strain 2835 with plasmid p96 and parent strain 2797 with parent plasmid p56. In plasmid p96, the parB locus was cloned in the forward orientation between AfeI and NotI restriction enzyme sites, thereby deleting a 271-bp nucleotide sequence from the plasmid backbone. Cell pellet titers of production strains 2797 and 2835 are shown.

[0077] Figure 5A -B. Fermentation titers of 4 new parB-containing production strains without any deletion of the plasmid backbone sequence. In strains 2884 (p117) and 2885 (p118), the parB locus was cloned in the AfeI restriction enzyme site in the For or Rev orientation, respectively. In strains 2886 (p119) and 2887 (p120), the parB locus was cloned in the ZraI restriction enzyme site in the For or Rev orientation, respectively. Cell clumps ( Figure 5A ) and culture medium ( Figure 5B ) potency.

[0078] Figure 6 . Map of plasmid p119 contained in strain 2886 showing the optimal position and orientation of the parB locus.

[0079] Figure 7 AB. Compatible two plasmids for testing the effects of periplasmic chaperone expression ( Figure 7 A) Compared with single plasmid ( Figure 7B) Schematic diagram of the expression system. In the two-plasmid system, the Fab vector contains the AmpR marker and the chaperone vector contains the KanR marker. For the single-plasmid system, the chaperone gene is cloned into a standard Fab vector with a KanR marker.

[0080] Figure 8 Fermentation titers of two strains expressing periplasmic chaperones (strains 2840 and 2841) and the control strain 2839 without any chaperone gene in a compatible two-plasmid expression system.

[0081] Fig.9A -B. Fermentation titers of two strains expressing periplasmic chaperone proteins (strain 2909 (p134) and strain 2910 (p135)) and parent strain 2886 (p119) in a single plasmid expression system. Cell clumps ( Fig.9A ) and culture medium ( Fig. 9B ) potency.

[0082] Fig.10 . Map of plasmid p134 contained in strain 2909 showing the location and orientation of the FkpA chaperone and parB loci.

[0083] Fig.11 .Cell pellet titers of various strains expressing the accessory factor parB locus and the FkpA chaperone protein in different locations and orientations in the plasmid.

[0084] Fig.12 AB. Fab heavy chain from strain 2886 captured in the Capto S column elution fraction ( Fig.12 A) and light chain ( Fig.12 B) Schematic representation of various translationally truncated (dashed arrows) and proteolytically trimmed (solid arrows) fragments. In this strain, typical intact Fab recovery was between 20%-25% of the total captured protein.

[0085] Fig.13A -B. Recoverable titers of various heavy chain (HC) carboxyl-terminal (C-terminal) extension variants ( Fig.13A ) and purity analysis ( Fig. 13B The new strains 2914 to 2918 (having plasmids p141 to p145, respectively) were compared with the control strain 2909 (having plasmid p134) without any HC C-terminal extension. Fig.13A Shown are the recoverable titers determined by Capto S column of heavy chain (HC) variants. Fig. 13B Shown is the purity analysis of the Capto S column pool of the heavy chain (HC) extension variants. DETAILED DESCRIPTION

[0086] Before describing the present invention in detail, it should be understood that the present invention is not limited to a particular method or composition or biological system which may of course vary. It should also be understood that the terms used herein are for the purpose of describing a particular embodiment only and are not intended to be limiting. Unless the content clearly provides otherwise, as used in this specification and the appended claims, the singular forms "a / an" and "the" include plural indicators. Thus, for example, reference to a "cell" includes combinations of two or more cells, etc.

[0087] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Those skilled in the art will now appreciate that many variations, changes, and substitutions do not depart from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein can be used to implement the present invention. It is intended that the following claims define the scope of the present invention, and methods and structures within the scope of these claims and their equivalents are covered by the claims.

[0088] Unless otherwise defined herein or in the rest of the specification below, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0089] definition

[0090] The term "amino acid" refers to naturally occurring and non-natural or unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) as well as pyrrolysine and selenocysteine. Amino acid analogs refer to compounds having the same basic chemical structure as naturally occurring amino acids, by way of example only, the α-carbon is bound to a hydrogen, a carboxyl group, an amino group, and a functional R group. The analogs may have modified R groups (for example, norleucine) or may have a modified peptide backbone, but still retain the same basic chemical structure as naturally occurring amino acids. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Amino acids may be referred to herein by their name, by their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Additionally, nucleotides may be referred to by their commonly accepted single-letter codes.

[0091] The term "antibody" herein refers to a protein consisting of one or more polypeptides that are substantially encoded by all or part of an antibody gene. Immunoglobulin genes include, but are not limited to, kappa, lambda, alpha, gamma (IgG1, IgG2, IgG3, and IgG4), delta, epsilon, and mu constant region genes, as well as tens of thousands of immunoglobulin variable region genes. In this article, antibodies are also intended to include full-length antibodies and antibody fragments, and include antibodies, antibody variants, engineered antibodies, and antibody fragments that naturally exist in any organism. In this article, antibodies are also intended to include complete antibodies, monoclonal antibodies, or polyclonal antibodies. In this article, antibodies also encompass multispecific antibodies and / or bispecific antibodies. The antibodies of the present invention include human antibodies. Human antibodies are generally composed of two light chains and two heavy chains, each comprising a variable region and a constant region. The light chain variable region comprises three CDRs flanked by a framework region, identified herein as CDRL1, CDRL2, and CDRL3. The heavy chain variable region comprises three CDRs flanked by a framework region, identified herein as CDRH1, CDRH2, and CDRH3.

[0092] The term "antibody fragment" refers to any form of an antibody other than the full-length form in this article. Antibody fragments include antibodies that are smaller components present in full-length antibodies, as well as engineered antibodies, such as antibody variants. Antibody fragments include, but are not limited to, Fv, Fc, Fab and (Fab')2, single-chain Fv (scFv), miniature bifunctional antibodies, miniature trifunctional antibodies, miniature tetrafunctional antibodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains and variable regions, and alternative skeleton non-antibody molecules, bispecific antibodies, etc. (Maynard and Georgiou, Annu. Rev. Biomed. Eng. 2: 339-76, 2000; Hudson, Curr. Opin. Biotechnol. 9: 395-402, 1998). Another functional substructure is a single-chain Fv (scFv), which comprises the variable regions of the heavy and light chains of immunoglobulins, covalently linked by a peptide linker (Hu et al., Cancer Research, 56, 3055-3061, 1996). These small (Mr 25,000) proteins typically retain specificity and affinity for antigens in the form of a single polypeptide, and can provide suitable building blocks for larger antigen-specific molecules. Unless otherwise specifically stated, statements and claims using the term "antibody" or "antibodies" specifically include "antibody fragments" and "antibody fragments". As used herein, the term "antibody fragment" when used in the context of an antibody refers to a portion of an antibody that is about 10% to about 99% of the length of a complete antibody. For example, a fragment of an antibody can be at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25% of the length of the intact antibody.A fragment of an antibody can also be at least about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, %, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, the antibody fragment is a diabody (Db), a BiTE (bispecific T cell engager), a DART (dual affinity retargeting) or a tandem diabody (TandAb).

[0093] In some embodiments, the disclosure relates to amino acids that have been biosynthetically incorporated into antibodies. As used herein, the term "biosynthetically" refers to any method utilizing a translation system (cellular or non-cellular), including the use of at least one of the following components: polynucleotides, codons, tRNAs, and ribosomes. For example, non-natural amino acids can be "biosynthetically incorporated" into non-natural amino acid polypeptides using the methods and techniques described herein and this is well known in the art. See, for example, WO2010 / 011735 and WO2005 / 074650.

[0094] As used herein, the terms "biologically active molecule," "biologically active moiety," or "biologically active agent" refer to any substance that can affect any physical or biochemical property of a biological system, pathway, molecule, or interaction associated with an organism, including, but not limited to, viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals, and humans. In particular, as used herein, biologically active molecules include, but are not limited to, any substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals, or otherwise enhancing the physical or mental health of humans or animals. Examples of biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, vaccines, immunogens, hard drugs, soft drugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxoids, biologically active molecules, prokaryotic and eukaryotic cells, viruses, polysaccharides, nucleic acids, and portions thereof, obtained or derived from viruses, bacteria, insects, animals, or any other cells or cell types, liposomes, microparticles, and micelles. Classes of bioactive agents suitable for use in the present invention include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, antifungals, bile acid resins, niacin and / or statins, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, antianxiety agents, hormones, growth factors, steroid agents, checkpoint protein inhibitors, signal transduction pathway inhibitors, bioactive molecules of microbial origin, etc. Bioactive agents also include amide compounds, such as those described in U.S. Patent Application Publication No. 20080221112.

[0095] As used herein for protein refolding, "reducing agent" is defined as any compound or material that maintains sulfhydryl groups in a reduced state and reduces intramolecular or intermolecular disulfide bonds. Suitable reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine (2-aminoethanethiol), and reduced glutathione. It is apparent to those of ordinary skill in the art that a variety of reducing agents are suitable for use in the methods and compositions of the present invention.

[0096] As used herein with respect to protein refolding, "oxidizing agent" is defined as any compound or material capable of removing electrons from an oxidized compound. Suitable oxidizing agents include, but are not limited to, oxidized glutathione, cystine, cystamine, oxidized dithiothreitol, oxidized erythritol, and oxygen. It is apparent to those of ordinary skill in the art that a variety of oxidizing agents are suitable for use in the methods of the present invention.

[0097] As used herein, "denaturing agent" or "denaturant" is defined as any compound or material that will cause the reversible unfolding of a protein. The strength of the denaturing agent or denaturant will be determined by the nature and concentration of the specific denaturing agent or denaturant. Suitable denaturing agents or denaturants can be chaotropes, detergents, organic solvents, water-miscible solvents, phospholipids, or a combination of two or more such agents. Suitable chaotropes include, but are not limited to, urea, guanidine, and sodium thiocyanate. Useful detergents may include, but are not limited to, strong detergents such as sodium dodecyl sulfate or polyoxyethylene ethers (e.g., Tween or Triton detergents), Sarkosyl, mild nonionic detergents (e.g., digitonin), mild cationic detergents (e.g., N->2,3-(diethoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents (e.g., sodium cholate or sodium deoxycholate), or zwitterionic detergents including, but not limited to, sulfobetaines (amphoteric detergents), 3-(3-chloroamidopropyl)dimethylammonio-1-propanesulfonate ( As denaturants, organic, water-miscible solvents (such as acetonitrile), lower alkanols (especially C2-C4 alkanols, such as ethanol or isopropanol) or lower alkanediols (especially C2-C4 alkanols, such as ethylene glycol) can be used. The phospholipids that can be used in the present invention can be naturally occurring phospholipids, such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine and phosphatidylinositol or synthetic phospholipid derivatives or variants, such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

[0098] As used herein, "refolding" describes any process, reaction, or method that converts a disulfide bond-containing polypeptide from an improperly folded or unfolded state with respect to the disulfide bonds to a native or correctly folded conformation.

[0099] As used herein, "cofolding" specifically refers to a refolding process, reaction or method that uses at least two polypeptides that interact with each other and results in the conversion of unfolded or improperly folded polypeptides into native, properly folded polypeptides.

[0100] "Conservatively modified variants" apply to amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, "conservatively modified variants" refer to those nucleic acids encoding the same or substantially the same amino acid sequence, or when the nucleic acid does not encode an amino acid sequence, it refers to substantially the same sequence. The term "conservatively modified variants" applies to natural and non-natural amino acids and natural and non-natural nucleic acid sequences and combinations thereof. With respect to a particular nucleic acid sequence, "conservatively modified variants" refer to those natural and non-natural nucleic acids encoding the same or substantially the same natural and non-natural amino acid sequence, or when natural and non-natural nucleic acids do not encode natural and non-natural amino acid sequences, it refers to substantially the same sequence. For example, due to the degeneracy of the genetic code, many functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position where alanine is specified by a codon, the codon can be changed to any corresponding codon described without changing the encoded polypeptide. The nucleic acid variation is a "silent variation" as a type of conservatively modified variation. Thus, for example, each natural or non-natural nucleic acid sequence encoding a natural or non-natural polypeptide herein also describes each possible silent variation of the natural or non-natural nucleic acid. One of ordinary skill in the art will recognize that each codon in a natural or non-natural nucleic acid (except AUG, which is usually the only codon for methionine, and UGG, which is usually the only codon for tryptophan) can be modified to obtain functionally identical molecules. Thus, each silent variation of natural and non-natural nucleic acids encoding natural and non-natural polypeptides is implicit in each described sequence. With regard to amino acid sequences, individual substitutions, deletions or additions of nucleic acids, peptides, polypeptides or protein sequences that change, add or delete a single natural and non-natural amino acid or a smaller percentage of natural and non-natural amino acids in the encoded sequence are "conservatively modified variants", wherein the changes result in the deletion of amino acids, the addition of amino acids or the substitution of natural and non-natural amino acids by chemically similar amino acids. Providing conservative substitution tables for functionally similar natural amino acids is well known in the art. Providing conservative substitution tables for functionally similar amino acids is known to those of ordinary skill in the art.The following eight groups each contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine ​​(C), Methionine (M) (see, e.g., Creighton, Proteins: Structures and Molecular Properties, WH Freeman & Co. 2nd Edition, 1993). Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the compositions described herein.

[0101] As used herein, the term "identical" refers to two or more sequences (nucleic acid or polypeptide) or identical subsequences. In addition, the term "substantially identical" as used herein refers to two or more sequences of sequence units with a certain percentage when compared and compared for maximum consistency on a comparison window or a specified region, as measured using a comparison algorithm or by manual comparison and visual inspection. For example only, if the continuous units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical or about 95% identical over a specified region, then two or more sequences may be "substantially identical". The percentage describes the "identity percentage" of two or more sequences. The identity of the sequence can be over a region of at least about 75-100 continuous units in length, over a region of about 50 continuous units in length, or across the entire sequence when not specified and present. This definition also relates to the complementary sequence of the test sequence. By way of example only, two or more polypeptide sequences are identical when the amino acid residues are identical over a specified region, while two or more polypeptide sequences are "substantially identical" if the amino acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical. The identity can exist over a region that is at least about 75 to about 100 amino acids in length, over a region that is about 50 amino acids in length, or, when not specified, across the entire sequence of a polypeptide sequence. In addition, by way of example only, two or more polynucleotide sequences are identical when the nucleic acid residues are identical over a specified region, and two or more polynucleotide sequences are "substantially identical" if the nucleic acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical. The identity can exist over a region of at least about 75 to about 100 nucleic acids in length, can exist over a region of about 50 nucleic acids in length, or, where not specified, spans the entire sequence of a polynucleotide sequence.

[0102] As used herein, the term "isolated" refers to the separation and removal of a target component from non-target components. When applied to nucleic acids or proteins, the term "isolated" refers to the nucleic acid or protein being free of at least some cellular components associated with it in the natural state, or the nucleic acid or protein having a concentration to a level higher than its in vivo or in vitro production concentration. The isolated material may be in a dry or semi-dry state, or in a solution, including but not limited to an aqueous solution. The isolated component may be in a homogeneous state, or the isolated component may be a part of a pharmaceutical composition comprising additional pharmaceutically acceptable carriers and / or excipients. Purity and homogeneity may be determined using analytical chemistry techniques including but not limited to polyacrylamide gel electrophoresis or high performance liquid chromatography. In addition, when the target component is isolated and is the main species present in the preparation, the component is described herein as being substantially purified. As used herein, the term "purified" may refer to a target component being at least 85% pure, at least 90% pure, at least 95% pure, at least 99% or greater percentage pure. By way of example only, a nucleic acid or protein is "isolated" when it is free of at least some of the cellular components with which it is associated in its native state, or when the nucleic acid or protein has been concentrated to a level greater than its in vivo or in vitro production concentration. Also, by way of example, a gene is isolated when it is separated from open reading frames that flank the gene and encode a protein other than the gene of interest.

[0103] As used herein, the term "modified" refers to any change made to a given polypeptide, such as a change in the length, amino acid sequence, chemical structure, co-translational modification or post-translational modification of the polypeptide. In some cases, the polypeptide is optionally modified, that is, the polypeptide may be modified or unmodified.

[0104] "Non-natural amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine. Other terms that can be used synonymously with the term "non-natural amino acid" are "non-naturally encoded amino acids", "non-natural amino acids", "non-naturally occurring amino acids" and their various forms of hyphenation and non-hyphenation. The term "non-natural amino acid" includes, but is not limited to, amino acids that exist naturally by modifying naturally encoded amino acids (including, but not limited to, 20 common amino acids or pyrrolysine and selenocysteine), but they themselves are not incorporated into the growing polypeptide chain by the translation complex. Examples of naturally occurring amino acids that are not naturally encoded include, but are not limited to, N-acetylglucosamine-L-serine, N-acetylglucosamine-L-threonine and O-phosphotyrosine. In addition, the term "non-natural amino acid" includes, but is not limited to, amino acids that do not exist naturally and can be obtained synthetically or can be obtained by modifying non-natural amino acids.

[0105] The term "nucleic acid" as used herein refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. By way of example only, the nucleic acids and nucleic acid polymers include, but are not limited to, (i) analogs of natural nucleotides that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides; (ii) oligonucleotide analogs, including but not limited to PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.); (iii) conservatively modified variants thereof (including but not limited to degenerate codon substitutions) as well as complementary sequences and explicitly indicated sequences. For example, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

[0106] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. That is, descriptions involving polypeptides are equally applicable to descriptions of peptides and to descriptions of proteins, and vice versa. The terms are applicable to naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are non-natural amino acids. In addition, such "polypeptides", "peptides" and "proteins" include amino acid chains of any length, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds. In an embodiment of the present invention, the target protein is a cytokine, a growth factor, a growth factor receptor, an interferon, an interleukin, an inflammatory molecule, an oncogene product, a peptide hormone, a signal transduction molecule, a steroid hormone receptor, a transcription activator, a transcription inhibitor, erythropoietin (EPO), insulin, human growth hormone, epithelial neutrophil activation peptide-78, GROα / MGSA, GROβ, GRO, MIP-1α, MIP-1, MCP-1, hepatocyte growth factor, insulin-like growth factor, leukemia inhibitory factor, oncostatin M, PD-ECSF , PDGF, pleiotropin, SCF, c-kit ligand, VEGF, G-CSF, IL-1, IL-2, IL-8, IGF-I, IGF-II, FGF (fibroblast growth factor), TNF, TGF-α, TGF-β, EGF (epidermal growth factor), KGF (keratinocyte growth factor), SCF / c-Kit, CD40L / CD40, VLA-4 / VCAM-1, ICAM-1 / LFA-1, hyaluronic acid / CD44, Mos, Ras, Raf , Met, p53, Tat, Fos, Myc, Jun, Myb, Rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and corticosterone, alpha-1 antitrypsin, angiostatin, antihemolytic factor, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic peptide, atrial peptide, CXC chemokine, T39765, NAP-2, ENA-78, Gro-a or Gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit ligand, cytokine , CC chemokine, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1α, monocyte inflammatory protein-1β, RANTES, I309, R83915, R91733, HCC1, T58847, D31065, T64262, C-kit ligand, collagen, colony stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activation peptide-78, epidermal growth factor (EGF), epithelial neutrophil activation peptide,Erythropoietin (EPO), shedding toxin, factor IX, factor VII, factor VIII, factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, G-CSF, GM-CSF, glucocerebrosidase, gonadotropin, growth factor, growth factor receptor, Hedgehog protein, hemoglobin, hepatocyte growth factor (HGF), hirudin, human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon, IFN-α, IFN-β, IFN-γ , interleukins, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurotrophin, neutrophil inhibitory factor (NIF), oncostatin M, osteogenic protein, oncogene products, parathyroid hormone, PD-ECSF, PDGF, peptide hormones, human growth hormone, pleiotrophin, protein A, protein G, pyrogenic exotoxin A, B or C, relaxin, renin, SCF, soluble complement receptor I, soluble I-CAM 1. Soluble interleukin receptor, soluble TNF receptor, somatomedin, somatostatin, growth hormone, streptokinase, superantigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin α1, tissue plasminogen activator, tumor growth factor (TGF), TGF-α, TGF-β, tumor necrosis factor, tumor necrosis factor α, tumor necrosis factor β, tumor necrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular endothelial growth factor (VEGF), urokinase, Mos, Ras, Raf, Met, p53, Tat, Fos, Myc, Jun, Myb, Rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and corticosterone, etc.

[0107] The term "post-translational modification" refers to any modification of a natural or unnatural amino acid that occurs after such amino acid has been translated and incorporated into a polypeptide chain. Such modifications include, but are not limited to, co-translational modifications in vivo, co-translational modifications in vitro (e.g., in a cell-free translation system), post-translational modifications in vivo, and post-translational modifications in vitro.

[0108] The term "recombinant host cell", also referred to as "host cell", refers to a cell containing an exogenous polynucleotide, wherein the method for inserting the exogenous polynucleotide into the cell includes, but is not limited to, direct uptake, transduction, f-mating, or other methods known in the art to produce a recombinant host cell. By way of example only, such an exogenous polynucleotide may be a non-integrating vector, including but not limited to a plasmid, or may be integrated into the host genome.

[0109] As used herein, the term "medium" includes any culture medium, solution, solid, semisolid or rigid support that can support or contain any host cell, including bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, Escherichia coli or Pseudomonas host cells and cell contents. Thus, the term can encompass culture media in which host cells have been grown, such as culture media in which target proteins have been secreted, including culture media before or after the proliferation step. The term can also encompass buffers or reagents containing host cell lysates, such as when the target protein is produced intracellularly and the host cells are dissolved or destroyed to release the target protein.

[0110] As used herein, the term "substantially purified" refers to a target component that is substantially or substantially free of other components that typically accompany or interact with the component of interest prior to purification. By way of example only, a target component may be "substantially purified" when its preparation contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating components. Thus, a "substantially purified" target component may have a purity level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or more. By way of example only, a natural amino acid polypeptide or a non-natural amino acid polypeptide may be purified from a natural cell, or from a host cell in the case of a recombinantly produced natural amino acid polypeptide or a non-natural amino acid polypeptide. For example, a preparation of a natural amino acid polypeptide or a non-natural amino acid polypeptide may be "substantially purified" when the preparation contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating material. For example, when a natural amino acid polypeptide or a non-natural amino acid polypeptide is recombinantly produced by a host cell, the natural amino acid polypeptide or the non-natural amino acid polypeptide may be present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cells. For example, when the natural amino acid polypeptide or non-natural amino acid polypeptide is recombinantly produced by host cells, the natural amino acid polypeptide or non-natural amino acid polypeptide may be present in the culture medium at about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L, about 100 mg / L, about 50 mg / L, about 10 mg / L, or about 1 mg / L or less of dry weight of cells. For example, a "substantially purified" natural amino acid polypeptide or non-natural amino acid polypeptide can have a purity level of about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more, as determined by an appropriate method, including but not limited to SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0111] In the present invention, the target protein, peptide, polypeptide to be expressed can be any protein, peptide, polypeptide expressed in E. coli in an unstable form and / or insoluble form or in a soluble form. In some embodiments, the insoluble protein can be any protein, peptide, polypeptide in E. coli located in the cytoplasm. In other aspects of the present invention, the soluble protein can be any protein, peptide, polypeptide located in the periplasm or cytoplasm or extracellular medium in E. coli. In some embodiments, such proteins, peptides, polypeptides include therapeutic, preventive or diagnostic proteins, peptides, polypeptides. In other embodiments, such proteins, peptides, polypeptides include interferons, interleukins, interleukin receptors, interleukin receptor antagonists, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor, erythropoietin, thrombopoietin, leukemia inhibitors, stem cell growth factor, tumor necrosis factor, growth hormone, proinsulin, insulin-like growth factor, fibroblast growth factor, platelet-derived growth factor, transforming growth factor, hepatocyte growth factor, bone morphogenetic factor, nerve growth factor, ciliary nerve growth factor, trophic factor, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, neurotrophin, pro-urokinase, tissue plasminogen activator, coagulation factor, protein C, glucocerebrosidase, superoxide dismutase, renin, lysozyme, P450, prorennin, trypsin inhibitor, elastase inhibitor, lipocortin, reptin, immunoglobulin, single-chain antibody, complement components, serum albumin, cedar pollen allergen, hypoxia-induced stress protein, protein kinase, proto-oncogene products, transcription factors and viral constitutive proteins, but are not limited thereto.

[0112] The term "derived from" refers to a component isolated from an organism, or a component produced by separation and modification, or, for example, chemical synthesis, using information from a component from an organism.

[0113] The term "translation system" refers to the components necessary to incorporate naturally occurring amino acids into a growing polypeptide chain (protein). For example, the components may include ribosomes, tRNA, synthetases, mRNA, etc. The components of the present invention may be added to a translation system in vivo or in vitro.

[0114] The term "prokaryotic organism" refers to a non-eukaryotic organism belonging to the Eubacteria (including but not limited to Escherichia coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, etc.), phylogenetic domain, or Archaea (including but not limited to Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium such as Halobacterium vorticella and Halobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Thermococcus horikoshii, Archaea thermophilus, etc.) phylogenetic domain.

[0115] The present invention provides an "operon" encoding Fab. The term "operon" used in the present invention is defined as a group of genes, each of which encodes the above-mentioned Fab heavy chain and light chain, forming a transcription unit under the control of a single promoter, and the operon includes a natural or artificial operon. In some embodiments of the present invention, the operon is a bicistronic operon driven by the Escherichia coli alkaline phosphatase (phoA) promoter.

[0116] As used herein, the term "promoter" refers to a nucleic acid sequence to which a regulatory nucleic acid is operably linked. Such promoters are known to be cis-acting sequence elements required for transcription because they act to bind to DNA-dependent RNA polymerases that transcribe sequences present downstream thereof. In order to regulate the expression level of the target protein (or gene) of the present invention, it is preferred that the promoter controlling transcription is an inducible promoter. Examples of inducible promoters include, for example, lac, tac, trc, trp, araB, Pzt-1, λP L etc. The lac, tac and trc promoters can be induced by using isopropyl-1-thio-β-D-galactopyranoside (IPTG); the trp, araB and Pzt-1 promoters can be induced by using 3-indole acrylic acid (IAA), L-arabinose and tetracycline, respectively; and the εPL promoter can be induced at high temperature (42°C). The T7 promoter, which is specifically and strongly transcribed by the T7 RNA polymerase, can also be used. In the transcription by the T7 RNA polymerase, an Escherichia coli strain carrying a lysed λ phage carrying the T7 RNA polymerase gene located downstream of the lac promoter is used, making it possible to induce the above-mentioned T7 RNA polymerase by using IPTG. In some embodiments, the inducible promoter is an inducible tetracycline promoter (pTc). In some embodiments of the present invention, the inducible promoter used to express genetic elements such as chaperone proteins may be the same as or different from the promoter used to express the target protein. In some embodiments, it may be advantageous to independently control the expression of genetic elements such as chaperones and the expression of desired proteins, where the level and timing of chaperone expression is optimized without reducing the expression level of the desired protein.

[0117] In other embodiments, the term "promoter" refers to a DNA region that is usually located upstream (towards the 5' region of the gene) required for transcription. Promoters allow the genes they control to be properly activated or repressed. Promoters contain specific sequences recognized by transcription factors. These factors bind to the promoter DNA sequence and cause the recruitment of RNA polymerase, which is an enzyme that synthesizes RNA from the gene coding region. In prokaryotes, promoters are recognized by RNA polymerase and related σ factors, and then brought into the promoter DNA species by activating proteins that bind to nearby self DNA sequences. In eukaryotes, the process is more complicated. For example, the transcription of RNA polymerase II promoters requires at least seven different factors. Promoter representatives can work in conjunction with other regulatory regions (enhancers, silencers, boundary elements / insulators) to guide the transcription level of a given gene. Promoters that can be used to implement the methods described herein include RNA polymerase III (also known as Pol III) promoters, which transcribe DNA to synthesize ribosomal 5S rRNA, tRNA and other small RNAs. Pol III is unusual (compared to Pol II) because it does not require a control sequence upstream of the gene. On the contrary, it can rely on internal control sequences. RNA polymerase III promoters are more variable in structure than the uniform RNA polymerase I promoters, but are not as diverse as RNA polymerase II promoters. They have been classified into three major types (types 1-3), two of which are intragenic and generally do not contain TATA, and one of which is extragenic and contains a TATA box.

[0118] As used herein, the term "encoding" is an open term, so that the nucleic acid encoding a specific amino acid sequence can be composed of codons for a specified polypeptide, or can also include another translatable sequence, or its presence is useful for controlling transcription, translation or replication, or promoting the operation of some host nucleic acid constructs. As used herein, the term is interpreted broadly and can have multiple applications. In some cases, the term "encoding" describes the process of semi-conservative DNA replication, in which one chain of a double-stranded DNA molecule is used as a template to encode a newly synthesized complementary sister chain by a DNA-dependent DNA polymerase. When used to describe the translation process, the term "encoding" is also extended to triplet codons encoding amino acids. In some cases, RNA molecules can encode DNA molecules, for example, by incorporating a reverse transcription process of an RNA-dependent DNA polymerase. On the other hand, DNA molecules can encode peptides, wherein it should be understood that the "encoding" used in this case combines both transcription and translation processes. On the other hand, the term "encoding" refers to any process, in which the information in one molecule is used to guide the generation of a second molecule whose chemical properties are different from the first molecule.

[0119] As used herein, the term "gene expression" refers to a process by which the coded information of a nucleic acid transcription unit (including, for example, genomic DNA or cDNA) is converted into an operable, inoperable or structural part of a cell, typically including the synthesis of a protein. Gene expression can be affected by external signals; for example, cells, tissues or subjects are exposed to agents that increase or decrease gene expression. The expression of a gene can also be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression is, for example, by acting on the control of transcription, translation, RNA transport and processing, degradation of intermediate molecules (such as mRNA), or by activation, inactivation, compartmentalization or degradation after the manufacture of a specific protein molecule, or by a combination thereof. Gene expression can be measured at the RNA level or protein level and by any method known in the art, including but not limited to Northern blotting, RT-PCR, Western blotting or in vitro, in situ or in vivo protein activity assays.

[0120] As used herein, the term "isolated" refers to a biological component (such as a nucleic acid molecule, peptide or cell) that has been purified from other biological components in a mixed sample (such as a cell extract). For example, an "isolated" peptide or nucleic acid molecule is a peptide or nucleic acid molecule that has been separated from other components of the cell in which the peptide or nucleic acid molecule is present (such as a host cell expressing a recombinant peptide or nucleic acid molecule).

[0121] As used herein, the terms "nucleic acid", "nucleic acid molecule", "polynucleotide" refer to a polymeric form of nucleotides, which may include sense and antisense strands of RNA, cDNA, genomic DNA and synthetic forms, and mixed polymers of the above. Nucleotide refers to a ribonucleotide, a deoxynucleotide or a modified form of either type of nucleotide. As used herein, "nucleic acid molecule" is synonymous with "nucleic acid" and "polynucleotide". Unless otherwise specified, a nucleic acid molecule is generally at least 10 bases in length. The term includes single-stranded and double-stranded forms of DNA. Nucleic acid molecules may include one or both of naturally occurring nucleotides and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages. As those skilled in the art will appreciate, As will be readily appreciated by those skilled in the art, nucleic acid molecules may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases. Such modifications include, for example, labeling, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), side groups (e.g., peptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylating agents, and modified linkages (e.g., α-anomeric nucleic acids, etc.). The term "nucleic acid molecule" also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpin, circular, and padlocked conformations.

[0122] As used herein, the term "prokaryote" or "prokaryotic" refers to organisms belonging to the kingdom Prokaryotes (also known as Procarya). In general, prokaryotes are distinguished from eukaryotic organisms by their unicellular organization, asexual reproduction by budding or fission, lack of a membrane-bound nucleus or other membrane-bound organelles, circular chromosomes, presence of operons, absence of introns, message caps, and poly-A mRNA and other biochemical characteristics, such as distinguished ribosomal structures. Prokarya includes the subkingdoms Eubacteria and Archaea (sometimes referred to as "primitive bacteria"). Cyanobacteria (blue-green algae) and mycoplasmas are sometimes divided into different categories under the kingdom Prokaryotes.

[0123] As used herein, the term "transduction" refers to the process of inserting genetic material, such as DNA or other nucleic acid molecules, into a cell. Common transduction techniques include the use of viral vectors (including bacteriophages), electroporation, and chemicals that increase cell permeability. Transfection and transformation are other terms for transduction, although these sometimes also imply expression of genetic material.

[0124] The term "transformation", "converted" or "introducing nucleic acid into a host cell" means that an extracellular nucleic acid (exogenous or heterologous DNA) such as a vector (with or without accompanying substances) enters a cell (e.g., a host cell). The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. For example, in prokaryotic cells, the transforming DNA may be maintained on an additional element such as a vector or a plasmid. The term "cell transformation" or "converted cell" refers to a cell or its progeny into which an extracellular nucleic acid has been introduced and thus has an extracellular nucleic acid. Nucleic acid can be introduced into a cell so that the nucleic acid can be replicated as a chromosomal integrant or as an additional chromosomal element. Transformation of a suitable host cell with, for example, an expression vector can be accomplished by well-known methods such as microinjection, electroporation, particle bombardment or by chemical methods, such as calcium phosphate-mediated transformation, for example, described in Maniatis et al., Molecular Cloning, A laboratory Manual, Cold Spring Harbor Laboratory, 1982 or in Ausubel et al., Current protocols in molecular biology, John Wiley and Sons, 1994.

[0125] "Recombinant DNA technology" refers to the technology of combining two heterologous DNA molecules, usually due to in vitro ligation of DNA from different organisms. Recombinant DNA molecules are usually produced by experiments in genetic engineering. Synonyms include "genetic splicing", "molecular cloning" and "genetic engineering". The products of these operations produce "recombinants" or "recombinant molecules". As used herein, the terms "recombinant protein" or "recombinant polypeptide" refer to protein molecules expressed from recombinant DNA molecules.

[0126] The term vector or construct as used herein refers to a nucleic acid molecule capable of transporting a non-vector nucleic acid sequence introduced into a vector. One type of vector is a "plasmid", which refers to a circular double-stranded DNA to which a non-plasmid DNA fragment can be connected. Other vectors include cosmids, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs). Another type of vector is a viral vector, in which additional DNA segments can be connected to all or part of a viral genome. Some vectors can replicate autonomously in the host cell in which they are introduced (for example, a vector with a bacterial origin of replication replicates in a bacterial host). Other vectors can be integrated into the genome of the host cell after being introduced into the host cell, and replicated together with the host genome. Some vectors contain expression control sequences (such as promoters), and can instruct the transcription of an expressible nucleic acid sequence introduced into the vector, and such vectors are referred to as "expression vectors". The vector may also include one or more selectable marker genes and / or genetic elements known in the art.

[0127] The term "plasmid" as used herein refers to a DNA molecule that is separated from chromosomal DNA and can replicate autonomously. It is usually circular and double-stranded, and can occur naturally in bacteria, and sometimes naturally in eukaryotic organisms (e.g., 2 micron rings in Saccharomyces cerevisiae). The size of the plasmid can vary from about 1 to more than 400 kilobase pairs. Plasmids usually contain genes or gene cassettes that confer selective advantages to the bacteria (or other cells) carrying them, such as the ability to make bacteria (or other cells) have antibiotic resistance. Plasmids contain at least one DNA sequence that serves as a replication origin, which enables plasmid DNA to replicate independently of chromosomal DNA. The chromosomes of most bacteria are circular, but linear plasmids are also known. The plasmids used in genetic engineering are referred to as vectors. They can be used to transfer genes from one organism to another, and usually contain genetic markers that confer a phenotype that can be selected or targeted. Most also contain polylinkers or multiple cloning sites, which are short regions containing several commonly used restriction sites, thereby allowing easy insertion of DNA fragments at this position. In order to promote selection during transformation, the plasmid of the present invention may also contain a selective marker gene. The example of such selective marker gene includes ampicillin resistance gene, kanamycin resistance gene and chloramphenicol resistance gene. It is expected that the plasmid selective marker gene used is different from the selective marker gene (if any) contained in the chromosome of the strain. The plasmid as defined above can be introduced into the host by standard techniques. Regarding the transformation of prokaryotic host cells, see, for example, Cohen et al., Proc.Natl.Acad.Sci.USA 69,2110,1972 and Sambrook et al., Molecular Cloning, A Laboratory Manual, Supplement 3rd Edition Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 2001.

[0128] As used herein, the term "expression cassette" refers to a polynucleotide sequence encoding a polypeptide operably linked to a promoter and other transcriptional and translational control elements, including but not limited to enhancers, stop codons, internal ribosome entry sites, or polyadenylation sites. The cassette may also contain sequences that facilitate its movement from one host molecule to another.

[0129] As used herein, the term "expression construct" refers to an expression module or expression cassette consisting of a recombinant or synthetic DNA molecule containing the desired coding sequence and the appropriate nucleic acid sequence required for expressing the operably linked coding sequence in a specific host organism. The nucleic acid sequences necessary for expression in prokaryotes generally include a promoter and a ribosome binding site, usually together with other sequences. Typically, an expression vector includes a transcription unit comprising a specific nucleic acid sequence to be transcribed that is operably linked to a promoter. The vector in the host can be, for example, an autonomous or self-replicating plasmid, a cosmid, a phage, a virus, or a retrovirus.

[0130] The terms "host", "host cell" and "recombinant host cell" are used interchangeably herein to refer to a prokaryotic cell into which one or more vectors or isolated and purified nucleic acid sequences of the present invention have been introduced. It should be understood that such terms refer not only to the specific subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in the next generation due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term as used herein.

[0131] Genetic element or component

[0132] As used herein, "genetic elements" or "genetic elements" may include any molecule that can promote, enhance, strengthen, or act in concert with an auxiliary protein or factor to promote protein expression. A genetic element may promote transcription of one or more genes associated with or involved in protein expression. Such elements may be from non-eukaryotic or eukaryotic sources.

[0133] The present invention utilizes one or more genetic elements involved in protein expression, including difficult to express (DTE) proteins, protein folding and retention and stabilization of plasmids in prokaryotic systems. Such genetic elements include, but are not limited to, DNA coding sequences and auxiliary proteins and / or factors, including chaperones, folding enzymes and isomerases. In some embodiments of the present invention, such genetic elements include the presence of periplasmic chaperones, heavy chain (HC) carboxyl terminal extensions and plasmid distribution site parB. Many categories of auxiliary proteins and / or factors in Escherichia coli contribute to the production of correctly folded and active proteins. For example, chaperones bind to misfolded or unfolded proteins and contribute to correct folding. Other auxiliary proteins play a role in disulfide bond reduction / oxidation or isomerization. Peptidyl-prolyl isomerase participates in cis / trans peptidyl-prolyl bond interconversion, and proteases have the function of degrading misfolded or aggregated proteins.

[0134] It has been demonstrated in the art that overexpression of various classes of chaperone proteins in E. coli increases the titer of heterologous proteins (including antibody fragments) (Thomas et al., Applied Biochemistry and Biotechnology 66, 197, 1997; Kolaj et al., Microbial Cell Factories, 2009; Merdanovic et al., Annu. Rev. Microbiol. 65, 149, 2011). It is also known to those skilled in the art that antibody heavy chain (HC) polypeptides are difficult to fold, and peptidyl prolyl isomerization has been proposed as the rate-limiting step of HC folding (Feige et al., Mol. Cell 34, 569, 2009). In addition, it is difficult to achieve prokaryotic expression of antibodies or antibody fragments incorporating non-natural amino acids at selected sites.

[0135] The present invention utilizes accessory proteins and / or factors or genetic elements in the design and engineering of vectors and plasmids produced therefrom to achieve an increase in the titer of proteins incorporating unnatural amino acids.

[0136] It is well known in the art that the periplasmic localization of several auxiliary proteins and / or factors (including protein folding factors and chaperone proteins) catalyzes the correct assembly and folding of functional antibody fragments. For example, molecular chaperones promote appropriate isomerization and cell targeting by transient interactions with folding intermediates; and folding enzymes accelerate the rate-limiting steps along the folding path. It is also well known in the art that Escherichia coli is one of the most widely used hosts for producing recombinant proteins. Escherichia coli ideally serves as a host for producing heterologous proteins at low cost and high yield, because it can easily grow to high density, and the research on host-vector systems has always been the most advanced, and many high expression vectors have been developed. Therefore, the Escherichia coli host-vector system is most widely used as an expression system for heterologous genes.

[0137] The periplasm of Escherichia coli provides a more oxidizing environment than the cytosol, thereby promoting disulfide bond formation, and the periplasmic space also contains less host proteins compared to the cytosol, thereby promoting subsequent purification processes. However, there are usually problems when recovering a large amount of correctly folded protein. For example, the high expression of proteins (such as antibody fragments) increases the demand for protein folding, thereby promoting uncharacterized metabolic burdens on cells, thereby causing protein misfolding and aggregation. The periplasmic localization of several protein folding factors and chaperones can catalyze the correct assembly and folding of functional proteins (such as antibody fragments). A method for solving the problem of low to poor protein yield is to secrete recombinant proteins into the periplasmic space or culture medium. Therefore, in order to increase, improve and / or optimize protein yield, periplasmic protein folding factors in Escherichia coli can be utilized.

[0138] Another method for improving low to poor protein yield is to utilize auxiliary proteins and / or factors that participate in restoring the solubility and function of the recombinant protein product and affect cell metabolism in the periplasmic antibody fragment expression process, including protein folding factors, such as chaperones. Chaperones of the present invention can be any protein involved in protein folding, including but not limited to chaperones derived from Escherichia coli. Examples of such chaperones include, for example, DnaK, DnaJ, GrpE, GroEL, GroES, HscA / Hsc66, ClpA, ClpB, ClpS, ClpX, HtpG, HdeA, HdeB, HtpG, HslO, HscCosmY, SecB, Spy, Tig, etc. Other chaperones include periplasmic chaperones Skp, FkpA, SurA, PPiA, PPiD, but are not limited thereto. In some embodiments of the present invention, one or more chaperones can be used in combination. The co-expression of one or more auxiliary proteins and / or factors (such as chaperones) with the target protein can increase the level of soluble protein. In one embodiment, the one or more proteins capable of promoting protein folding are FkpA, Skp, SurA, PPiA and PPiD. In other embodiments, the one or more proteins capable of promoting protein secretion or translocation are SecY, SecE, SecG, SecYEG, SecA, SecB, FtsY and Lep. In another embodiment, the one or more proteins capable of promoting disulfide bond formation are DsbA, DsbB, DsbD, DsbG. In some embodiments, the disulfide bond oxidation and isomerization components can be from eukaryotic sources, such as protein disulfide isomerase (PDI) from yeast or humans.

[0139] Periplasmic chaperones, including but not limited to Skp and FkpA, have been shown to improve the periplasmic production of antibody fragments (Hayhurst et al., Protein Expr. Purif., 15, 336-343, 1999; Hayhurst et al., J. Immunol. Meth., 276, 185-196, 2003; Padiolleau-Lefèvre et al., Immunol. Lett., 103, 39-44, 2006; Zhang, et al., Biotechniques, 35, 1032-1038, 2003; Bothmann et al., J. Biol. Chem., 275, 17100-17105, 2000; Ow et al., Microb. Cell Fact., 9, 22, 2010). In E. coli, FkpA and Skp are two well-known bifunctional periplasmic chaperones with peptidyl-prolyl isomerase activity. Skp and FkpA are molecular chaperones with the general ability to refold misfolded proteins and prevent them from aggregating into insoluble inclusion bodies in the periplasm. Skp is a 17-kDa homotrimeric protein that promotes the correct folding of newly synthesized outer membrane proteins and helps maintain their solubility; and FkpA is a 26-kDa homodimeric protein that exhibits chaperone activity and peptidyl-prolyl isomerase (PPlase) activity.

[0140] In some embodiments, proteins that can promote disulfide bond formation can be used, including disulfide bond oxidation and isomerization chaperones and enzymes. Protein disulfide isomerases are enzymes that catalyze the formation and cleavage of disulfide bonds between cysteine ​​residues within proteins during protein folding. In E. coli, such disulfide isomerases (auxiliary proteins) include DsbA, DsbB, DsbC, DsbD, or DsbG. Co-expression of proteins that catalyze disulfide bond formation to improve protein expression in host cells is well known to those skilled in the art. For example, WO1998 / 56930 discloses a method for producing a polypeptide containing a heterologous disulfide bond in a bacterial cell, wherein a prokaryotic disulfide isomerase such as DsbC or DsbG is co-expressed with a eukaryotic polypeptide; U.S. Pat. No. 6673569 discloses an artificial operon comprising a polynucleotide encoding each of DsbA, DsbB, DsbC and DsbD for producing exogenous proteins; EP Patent No. 0786009 discloses a method for producing a heterologous polypeptide in bacteria, wherein the expression of a nucleic acid encoding DsbA or DsbC is induced before inducing the expression of a nucleic acid encoding the heterologous polypeptide. In addition, some protein isomerases (e.g., DsbC) are bifunctional and are essential in protein expression, as observed in the art for FkpA.

[0141] It is known in the art that in some bacterial plasmid separation systems, a specific gene called the plasmid partition site (par) is involved in killing daughter cells that lose plasmids during cell division (Ebersbach G. and Gerdes K., Annu. Rev. Genet. 2005 39, 453). In addition, other findings in the literature also show that the 580-bp minimum partition B (parB) site originally from Escherichia coli plasmid R1 mediates maintenance and stability in many bacteria (including Escherichia coli) (Gerdes K., Nature Biotech. 1988 6, 1402-1405; Gerdes K. et al., Mol. Microbiol. 19904, 1807; Gerdes K. and Neilsen Al, J. Mol. Biol. 1992 226, 637). The parB (partition B) locus of plasmid R1 controls plasmid stability and encodes hok and sok genes. Cells that lose the plasmid carrying parB during cell division are killed rapidly (so-called post-isolation killing; Gerdes et al., 1986a). The hok gene, which is responsible for host cell killing, is located to the left of the parB region. This gene is regulated by the repressor encoded by the sok gene, which is a small antisense RNA that is complementary to the hok mRNA at the horizontal level. The sok gene, which encodes a trans-acting antagonist of the activity of the hok gene (inhibits killing), is located just upstream of the entire gene coding frame (Gerdes et al., 1986a). The hok mRNA is very stable, while the sok RNA deteriorates rapidly. This mechanism of action, post-isolation killing, occurs due to the rapid disappearance of sok RNA molecules in cells that have lost the plasmid carrying parB. This in turn leads to the translation of the stable hok mRNA. Subsequently, the Hok protein is synthesized and promotes the killing of plasmid-free cells. Therefore, in order to improve plasmid retention in the production strains of the present disclosure, the parB locus gene is utilized in the design and engineering of synthetic expression plasmids. As observed in the examples herein, the parB locus is important in increasing the yield of recombinant protein expression.In some embodiments of the invention, the location and orientation of the parB locus with respect to the gene of interest also influences the increase in recombinant protein expression.

[0142] Protease plays an important role in transforming old and misfolded proteins in the periplasm and cytoplasm of Escherichia coli. Bacterial proteases play a role in degrading target recombinant proteins, thereby often significantly reducing the yield of active proteins. Such proteases include but are not limited to protease III (ptr), DegP, OmpT, Tsp, prlC, ptrA, ptrB, pepA-T, tsh, espc, eatA, clpP and ions. It is believed that periplasmic serine endoprotease Prc (processing for C-terminal) (also referred to as tail specific protease (Tsp)) is the cause of Fab cracking (Chen C. et al. Biotechnology and Bioengineering 2004 85,463). It is believed that Prc recognizes and binds to the C-terminal of protein, and then cracks in loose or unfolded regions within the protein sequence (Keiler et al. Protein Science 1995 4,1507). The cleavage site is determined by protein secondary or tertiary structure rather than primary sequence. Prc has a wide range of sequence specificity for proteolytic sites. It is known in the art that the C-terminal sequence of a protein can affect its cleavage by Prc, and that modifying the C-terminal sequence can change the amount of cleavage of a protein by Prc (Keiler KC and Sauer RT, The Journal of Biological Chemistry 1996 271, 2589). Therefore, in some embodiments, the present invention examines the involvement of proteases in regulating or modulating protein production.

[0143] Orthogonal tRNA technology

[0144] In optimizing protein expression in prokaryotic systems such as E. coli, the present invention utilizes proteins (including antibody fragments that are difficult to express (DTE)) into which non-naturally encoded amino acids are incorporated. The use of non-natural amino acid incorporation techniques in E. coli is well known in the art. This technology uses orthogonal tRNA / aminoacyl-tRNA synthetase pairs that function in prokaryotic species to specifically incorporate non-natural amino acids in response to selector codons. See, for example, WO 2002 / 085923, WO 2002 / 086075, WO 2004 / 09459, WO 2005 / 019415, WO 2005 / 007870, and WO 2005 / 007624, the contents of which are incorporated by reference in their entirety. See also, Wang and Schultz, "Expanding the Genetic Code," Angewandte Chemie Int. Ed., 44 (1): 34-66, 2005, by engineering a polynucleotide encoding a target protein (or gene) into a sequence containing a selector codon that marks the incorporation of an unnatural amino acid, the incorporation of an unnatural amino acid into a protein can be programmed to occur at any desired position. The selector codons of the present invention include, for example, unique three-base codons, nonsense codons, such as stop codons.

[0145] In order to add additional reactive non-natural amino acids to the genetic code, a new orthogonal pair comprising an aminoacyl-tRNA synthetase and a suitable tRNA is needed, which can effectively function in the host translation mechanism, but is "orthogonal" to the translation system in question, meaning that its function is independent of the synthetase and tRNA endogenous to the translation system. The desired characteristics of a straight homologue pair include a tRNA that only decodes or recognizes a specific codon (e.g., a selector codon) that is not decoded by any endogenous tRNA, and an aminoacyl-tRNA synthetase that preferentially aminoacylates (or "charges") the homologous tRNA with only one specific non-natural amino acid. O-tRNA is also generally not aminoacylated by endogenous synthetases. For example, in E. coli, an orthogonal pair will include an aminoacyl-tRNA synthetase that does not cross-react with any endogenous tRNA (e.g., there are 40 endogenous tRNAs in E. coli), and an orthogonal tRNA that is not aminoacylated by any endogenous synthetase (e.g., there are 21 endogenous synthetases in E. coli).

[0146] Various orthogonal tRNAs and aminoacyl tRNA synthetases have been described in the art for inserting specific synthetic amino acids into polypeptides, and are generally applicable to the present invention. For example, keto-specific O-tRNA / aminoacyl-tRNA synthetases are described in Wang et al., Proc.Natl.Acad.Sci.USA 100:56-61 (2003) and Zhang et al., Biochem.42 (22):6735-6746 (2003). Exemplary O-RS or a portion thereof is encoded by a polynucleotide sequence, and is included in the amino acid sequence disclosed in U.S. Patent Nos. 7,045,337 and 7,083,970, each of which is incorporated herein by reference. The corresponding O-tRNA molecules used with O-RS are also described in U.S. Patent Nos. 7,045,337 and 7,083,970, which are incorporated herein by reference. Additional examples of O-tRNA / aminoacyl-tRNA synthetase pairs are described in WO 2005 / 007870, WO 2005 / 007624 and WO 2005 / 019415.

[0147] Several other orthogonal pairs have been reported. Systems derived from glutaminyl (see, e.g., Liu et al., (1999) PNAS 96:4780-4785), aspartyl (see, e.g., Pastrnak et al., (2000) Helv. Chim. Acta 83:2277-2286) and tyrosyl (see, e.g., Ohno et al., (1998) J. Biochem. (Tokyo, Jpn.) 124:1065-1068; and Kowal et al., (2001) PNAS 98:2268-2273) of Saccharomyces cerevisiae tRNA and synthetase have been described for potential incorporation of unnatural amino acids into Escherichia coli. From E. coli glutaminyl (see, e.g., Kowal et al., (2001) PNAS 98:2268-2273) and tyrosyl (see, e.g., Edwards et al., (1990) Mol. Cell. Biol. 10:1633-1641) synthetase systems have been described for use in Saccharomyces cerevisiae. The E. coli tyrosyl system has been used to incorporate 3-iodo-L-tyrosine in vivo in mammalian cells. See Sakamoto et al., (2002) Nucleic Acids Res. 30:4692-4699.

[0148] Typically, when the orthogonal pair recognizes the selector codon and charges the amino acid in response to the selector codon, the orthogonal pair is referred to as "suppressing" the selector codon. That is, the selector codon that the endogenous machinery of the translation system (e.g., the cell) cannot recognize is generally not translated, which can result in blocking the production of a polypeptide that would otherwise be translated from the nucleic acid. The O-tRNA of the present invention recognizes the selector codon and includes at least about 45%, 50%, 60%, 75%, 80% or 90% or more of the inhibition efficiency in the presence of a cognate synthetase in response to the selector codon compared to the inhibition efficiency of the O-tRNA comprising or encoded by the polynucleotide sequence. The O-RS is an O-tRNA aminoacylated with a target non-natural amino acid. The cell uses an O-tRNA / O-RS pair to incorporate a non-natural amino acid into a growing polypeptide chain, for example, via a nucleic acid comprising a polynucleotide encoding a target polypeptide, wherein the polynucleotide comprises a selector codon recognized by the O-tRNA. In certain desirable aspects, the cell can comprise additional O-tRNA / O-RS pairs, wherein the additional O-tRNA is charged with a different unnatural amino acid through the additional O-RS. For example, one of the O-tRNAs can recognize a four-base codon and the other can recognize a stop codon. Alternatively, a plurality of different stop codons or a plurality of different four-base codons can specifically recognize different selector codons.

[0149] The selector codons of the present invention expand the genetic codon framework of the protein biosynthetic machinery. For example, selector codons include, but are not limited to, unique three-base codons, nonsense codons, such as stop codons, including but not limited to amber codons (UAG), ochre codons or opal codons (UGA), unnatural codons, four or more base codons, rare codons, etc. It is obvious to one of ordinary skill in the art that in a single polynucleotide encoding a target protein, a wide range of selector codons can be introduced into a desired gene or polynucleotide, including but not limited to one or more, two or more, three or more.

[0150] In one embodiment, the method involves the use of a selection factor codon (which is a stop codon) to incorporate one or more unnatural amino acids in vivo. For example, an O-tRNA is produced that recognizes a stop codon (including but not limited to UAG) and is aminoacylated by an O-RS with a desired unnatural amino acid. The aminoacyl-tRNA synthetase of a naturally occurring host does not recognize this O-tRNA. Conventional site-directed mutagenesis can be used to introduce a stop codon at the target site of a target polypeptide strain, including but not limited to TAG. See, for example, Sayers, JR, et al. (1988), 5'-3' Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res, 16: 791-802. When O-RS, O-tRNA, and a nucleic acid encoding a target polypeptide are combined in vivo, an unnatural amino acid is incorporated in response to a UAG codon to obtain a polypeptide containing an unnatural amino acid at a specified position.

[0151] Incorporation of unnatural amino acids in vivo can be accomplished without significant interference with eukaryotic host cells. For example, since the suppression efficiency of UAG codons depends on competition between O-tRNA (including but not limited to amber suppressor tRNA) and eukaryotic release factors (including but not limited to eRF) (which bind to the stop codon and initiate release of the growing peptide from the ribosome), the suppression efficiency can be regulated by (including but not limited to) increasing the expression level of O-tRNA and / or suppressor tRNA.

[0152] Unnatural amino acids can also be encoded with rare codons. For example, when reducing the concentration of arginine in an in vitro protein synthesis reaction, it has been shown that the rare arginine codon AGG is effective for inserting Ala by a synthetic tRNA acylated with alanine. See, for example, Ma et al., Biochemistry, 32:7939 (1993). In this case, the synthetic tRNA competes with the naturally occurring tRNAArg, which exists as a minor species in Escherichia coli. Some organisms do not use all triplet codons. The unassigned codon AGA in Micrococcus luteus has been used to insert amino acids in in vitro transcription / translation extracts. See, for example, Kowal and Oliver, Nucl. Acid. Res., 25:4685 (1997). The components of the present invention can be produced to use these rare codons in vivo.

[0153] Selector codons also include extended codons, including but not limited to four or more base codons, such as four bases, five bases, six bases or more base codons. Examples of four base codons include but are not limited to AGGA, CUAG, UAGA, CCCU, etc. Examples of five base codons include but are not limited to AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC, etc. Features of the present invention include the use of extended codons based on frameshift suppression. Four or more base codons can be inserted into the same protein with (including but not limited to) one or more non-natural amino acids. For example, in the presence of a mutant O-tRNA (including but not limited to a special frameshift suppressor gene tRNA) with an anticodon loop (e.g., with at least 8-10nt anticodon loops), four or more base codons are read as single amino acids. In other embodiments, the anticodon loop is decodable, including but not limited to at least four base codons, at least five base codons, or at least six base codons or more. Since there are 256 possible four base codons, four or more base codons can be used to encode a variety of non-natural amino acids in the same cell. See Anderson et al., Exploring the Limits of Codon and Anticodon Size, Chemistry and Biology, 9: 237-244.2002; Magliery, Expanding the Genetic Code: Selection of Efficient Suppressors of Four-base Codons and Identification of “Shifty” Four-base Codons with a Library Approach in Escherichia coli, J. Mol. Biol. 307:755-769, 2001.

[0154] For example, four-base codons have been used to incorporate unnatural amino acids into proteins using in vitro biosynthetic methods. See, e.g., Ma et al., Biochemistry, 32:7939, 1993; and Hohsaka et al., J. Am. Chem. Soc., 121(51), pp. 12194-12195, 1999. CGGG and AGGU were used to simultaneously incorporate 2-naphthylalanine and NBD derivatives of lysine into streptavidin in vitro by two chemically acylated frameshift suppressor tRNAs. (See, e.g., Hohsaka et al., supra). In an in vivo study, Moore et al. investigated the ability of tRNALeu derivatives with NCUA anticodons to suppress UAGN codons (N can be U, A, G, or C), and found that the quadruplex UAGA could be decoded by tRNALeu with UCUA anticodons with efficiencies of 13% to 26%, with almost no decoding in 0 or -1 frames. See Moore et al., J. Mol. Biol., 298:195, 2000. In one embodiment, extended codons based on rare codons or nonsense codons can be used in the present invention, which can reduce missense readthrough and frameshift suppression at other unwanted sites.

[0155] For a given system, the selector codon may also include one of the natural three-base codons, where the endogenous system does not use (or rarely uses) the natural base codon. For example, this includes systems that lack tRNAs that recognize natural three-base codons, and / or systems where the three-base codon is a rare codon.

[0156] The selection factor codon optionally includes an unnatural base pair. These unnatural base pairs further expand the existing genetic alphabet. An additional base pair increases the number of triplet codons from 64 to 125. The properties of the third base pair include stable and selective base pairing, efficient enzymatic incorporation into DNA with high fidelity by polymerase, and effective continuous primer extension after synthesizing the nascent unnatural base pair. Descriptions of unnatural base pairs applicable to methods and compositions include, for example, Hirao, et al., An unnatural base pair for incorporating amino acid analogues into protein, Nature Biotechnology, 20: 177-182, 2002. See also, Wu, Y., et al., J. Am. Chem. Soc. 124: 14626-14630, 2002. Other related publications are listed below.

[0157] In certain embodiments of the invention, cells such as Escherichia coli cells include orthogonal tRNA (O-tRNA), orthogonal aminoacyl-tRNA synthetase (O-RS), non-natural amino acids, and polynucleotides encoding target polypeptides, wherein the polynucleotides include selector codons recognized by O-tRNA. The translation system can also be a cell-free system, for example, any of a variety of commercially available "in vitro" transcription / translation systems. Examples of pairs include mutRNATyr-mutTyrRS pairs, such as mutRNATyr-SS12TyrRS pairs, mutRNALeu-mutLeuRS pairs, mutRNAThr-mutThrRS pairs, mutRNAGlu-mutGluRS pairs, etc.

[0158] Unnatural amino acids

[0159] The present disclosure includes antibody fragments with non-natural or non-naturally encoded amino acid sites specifically incorporated therein. Methods for site-specific introduction of non-natural amino acids in antibodies or antibody fragments are described in the art, see, for example, WO2010 / 011735 and WO2005 / 074650. Typically, the non-natural amino acids of the present invention are selected or designed to provide additional features that are not available in twenty kinds of natural amino acids. For example, non-natural amino acids are optionally designed or selected to modify the biological properties of the proteins into which they are incorporated. For example, by including non-natural amino acids in proteins, the following properties can be optionally modified: toxicity, biodistribution, solubility, stability (e.g., heat, hydrolysis, oxidation, resistance to enzymatic degradation, etc.), convenience of purification and processing, structural properties, spectral properties, chemical and / or photochemical properties, catalytic activity, redox potential, half-life, the ability to react with other molecules (covalently or non-covalently), etc.

[0160] As used herein, the term "non-natural or unnatural amino acid" refers to any amino acid, modified amino acid and / or amino acid analog that is not one of the 20 common naturally occurring amino acids or selenocysteine ​​or pyrrolysine. The non-natural amino acid of the present invention can be a naturally occurring compound other than the twenty α-amino acids known in the art. Unnatural amino acids may include, but are not limited to, p-ethylthiocarbonyl-L-phenylalanine, p-(3-oxobutyryl)-L-phenylalanine, 1,5-dansyl-alanine, 7-amino-coumarin amino acids, 7-hydroxy-coumarin amino acids, nitrobenzyl-serine, O-(2-nitrobenzyl)-L-tyrosine, p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridylalanine, p-(2-amino-1-hydroxyethyl)-L-phenylalanine, p-isopropylthiocarbonyl-L-phenylalanine, 3-nitro-L-tyrosine, and p-nitro-L-phenylalanine, and include the L and D enantiomers of such substances. The non-natural amino acids of the present invention can comprise alkyl-, aryl-, acyl-, hydrazine, cyano-, halide-, hydrazide, alkenyl, ether, borate, boronate ester, phosphate, phosphono, phosphine, enone, imine, ester, hydroxylamine, amine, etc., or any combination thereof. Other unnatural amino acids of interest include, but are not limited to, amino acids comprising a photoactivatable cross-linker, spin-labeled amino acids, fluorescent amino acids, metal binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that covalently or non-covalently interact with other molecules, photocaged amino acids and / or photoisomerizable amino acids, amino acids containing biotin or a biotin analog, keto-containing amino acids, glycosylated amino acids, sugar moieties attached to the side chains of amino acids, amino acids comprising polyethylene glycol or polyethers, heavy atom-substituted amino acids, chemically cleavable or photocleavable amino acids, amino acids with extended side chains compared to natural amino acids (e.g., polyethers or long chain hydrocarbons, greater than about 5, greater than about 10 carbons, etc.), carbon-linked sugar-containing amino acids, amino thioacid-containing amino acids, and amino acids containing one or more toxic moieties.

[0161] Non-natural amino acids based on natural amino acids, such as tyrosine, glutamine, phenylalanine, etc. are included in the present invention. Tyrosine analogs include para-substituted tyrosine, ortho-substituted tyrosine, and meta-substituted tyrosine, wherein the substituted tyrosine comprises acetyl, benzoyl, amino, hydrazine, hydroxylamine, thiol group, carboxyl, isopropyl, methyl, C6-C20 straight or branched hydrocarbon, saturated or unsaturated hydrocarbon, O-methyl, polyether group, nitro, etc. In addition, polysubstituted aromatic rings are also considered. Glutamine analogs of the present invention include but are not limited to α-hydroxy derivatives, β-substituted derivatives, cyclic derivatives, and amide-substituted glutamine derivatives. Exemplary phenylalanine analogs include but are not limited to meta-substituted phenylalanine, wherein the substituents include hydroxyl, methoxy, methyl, allyl, acetyl, etc. Specific examples of unnatural amino acids include, but are not limited to, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine and isopropyl-L-phenylalanine, and the like.

[0162] Many of the non-natural amino acids provided above are commercially available, for example, from Sigma (USA) or Aldrich (Milwaukee, Wis., USA). Those that are not commercially available can be optionally synthesized as provided in various publications or using standard methods known to those skilled in the art. For organic synthesis techniques, see, for example, Fessenden, R and Fessenden, J, Organic Chemistry, Second Edition, Willard Grant Press, Boston Mass., 1982; March, J., Advanced Organic Chemistry, Third Edition, Wiley and Sons, New York, 1985; and Carey, F. and Sundberg, R., Advanced Organic Chemistry, Third Edition, Parts A and B, Plenum Press, New York, 1990). Additional publications describing the synthesis of unnatural amino acids include, for example, WO 2002 / 085923 entitled "Invivo incorporation of Unnatural Amino Acids"; Matsoukas et al., J. Med. Chem., 38, 4660-4669, 1995; King, FE and Kidd, DAA, A New Synthesis of Glutamine and of gamma-Dipeptides of Glutamic Acid from Phthylated Intermediates, J. Chem. Soc., 3315-3319, 1949; Friedman, OM and Chatterrji, R., Synthesis of Derivatives of Glutamine as Model Substrates for Anti-Tumor Agents, J. Am. Chem. Soc. 81, 3750-3752, 1959; Craig, JC et al., Absolute Configuration of the Enantiomers of 7-Chloro-4[[4-(diethylamino)-1-methylbutyl]amino]quinoline (Chloroquine), J. Org. Chem. 53, 1167-1170, 1988; Azoulay, M., Vilmont, M. and Frappier, F.,Glutamine analogues as Potential Antimalarials,Eur.J.Med.Chem.26,201-5,1991; Koskinen,A.M.P. and Rapoport,H.Synthesis of 4-Substituted Prolines as Conformationally Constrained Amino Acid Analogues,J.Org.Chem.,54,1859-1866,1989; Christie,B.D. and Rapoport,H.,Synthesis of Optically Pure Pipecolates from L-Asparagine.Application to the Total Synthesis of(+)-Apovincamine through Amino Acid Decarbonylation and Iminium Ion Cyclization,J.Org.Chem.1989:1859-1866,1985; Barton et al.,Synthesis of Novel a-Amino-Acids and Derivatives Using Radical Chemistry:Synthesis of L-and D-a-Amino-Adipic Acids,L-a-aminopimelic Acid and Appropriate Unsaturated Derivatives,Tetrahedron Lett,43:4297-4308,1987; and Subasinghe et al.,Quisqualic acid analogues:synthesis of beta-heterocyclic 2-aminopropanoic acid derivatives and their activity at a novel quisqualate-sensitized site,J.Med.Chem.35:4602-7,1992.See also International Publication WO 2004 / 058946,entitled "Protein Arrays",filed Dec.22,2003.

[0163] The incorporation of unnatural amino acids can be performed for a variety of purposes, including but not limited to modulating the interaction of a protein with its receptor or one or more subunits of its receptor, adjusting changes in protein structure and / or function, changing size, acidity, nucleophilicity, hydrogen bonding, hydrophobicity, accessibility to protease target sites, targeting to a certain part (including but not limited to protein arrays), adding biologically active molecules, attaching polymers, attaching radionuclides, modulating serum half-life, modulating tissue permeability (e.g., tumors), modulating active transport, modulating tissue, cell or organ specificity or distribution, modulating immunogenicity, modulating protease resistance, etc. Proteins containing unnatural amino acids can have enhanced or even completely new catalytic or biophysical properties. For example, the following properties are modified by including unnatural amino acids in proteins: receptor binding, toxicity, biodistribution, structural properties, spectral properties, chemical and / or photochemical properties, catalytic ability, half-life (including but not limited to serum half-life), ability to react with other molecules (including but not limited to covalently or non-covalently), etc. Compositions comprising proteins containing at least one unnatural amino acid can be used for (including but not limited to) novel therapeutic agents, diagnostic agents, catalytic enzymes, industrial enzymes, binding proteins (including but not limited to antibodies), and (including but not limited to) studies of protein structure and function. See, for example, Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structure and Function, Current Opinion in Chemical Biology, 4: 645-652.

[0164] In one aspect of the invention, a composition comprises at least one protein having at least one, including but not limited to at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more unnatural amino acids. The unnatural amino acids may be the same or different, including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different sites in the protein, the sites comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different unnatural amino acids. On the other hand, the composition comprises a protein, at least one, but less than all, of the specific amino acids present in the protein are substituted with unnatural amino acids. For a given protein having more than one unnatural amino acid, the unnatural amino acids may be the same or different (including but not limited to, the protein may comprise two or more different types of unnatural amino acids, or may comprise two of the same unnatural amino acids). For a given protein having more than two unnatural amino acids, the unnatural amino acids may be the same, different, or a combination of multiple unnatural amino acids of the same type and at least one different unnatural amino acid.

[0165] The target protein or polypeptide with at least one non-natural amino acid is a feature of the present invention. The present invention also includes polypeptides or proteins with at least one non-natural amino acid produced using the compositions and methods of the present invention. Excipients (including but not limited to pharmaceutically acceptable excipients) may also be present with the protein. By producing a target protein or polypeptide with at least one non-natural amino acid in a eukaryotic cell, the protein or polypeptide will generally comprise a eukaryotic post-translational modification. In certain embodiments, the protein comprises at least one non-natural amino acid and at least one post-translational modification performed in vivo by a eukaryotic cell, wherein the post-translational modification is not performed by a prokaryotic cell. For example, the post-translational modification includes but is not limited to acetylation, acylation, lipid modification, palmitoylation, palmitate addition, phosphorylation, glycolipid linkage modification, glycosylation, etc.

[0166] One advantage of non-natural amino acids is that they have additional chemical moieties that can be used to add other molecules. These modifications can be performed in vivo or in vitro in eukaryotic or non-eukaryotic cells. Therefore, in certain embodiments, post-translational modifications are performed by non-natural amino acids. For example, post-translational modifications can be performed by nucleophilic-electrophilic reactions. Most reactions currently used for selective modification of proteins involve covalent bond formation between nucleophilic and electrophilic reaction partners, including but not limited to the reaction of -haloketones with histidine or cysteine ​​side chains. In these cases, selectivity depends on the number and accessibility of nucleophilic residues in the protein. In the proteins of the present invention, other more selective reactions can be used, such as the reaction of non-natural keto-amino acids with hydrazides or aminooxy compounds in vitro and in vivo. See, for example, Cornish et al., (1996) J.Am.Chem.Soc., 118: 8150-8151; Mahal et al., (1997) Science, 276: 1125-1128; Wang et al., (2001) Science 292:498-500; Chin et al., (2002) J. Am. Chem. Soc. 124:9026-9027; Chin et al., (2002) Proc. Natl. Acad. Sci., 99:11020-11024; Wang et al., (2003) Proc. Natl. Acad. Sci., 100:56-61; Zhang et al., (2003) Biochemistry, 42:6735-6746; and Chin et al., (2003) Science, 301:964-7, all of which are incorporated herein by reference. This allows for the selective labeling of virtually any protein with a variety of reagents, including fluorophores, cross-linking agents, sugar derivatives, and cytotoxic molecules. See U.S. Pat. No. 6,927,042, entitled "Glycoprotein synthesis," which is incorporated herein by reference. Post-translational modifications (including but not limited to by azido amino acids) can also be carried out by Staudinger ligation (including but not limited to, using triarylphosphine reagents). See, e.g., Kiick et al., Incorporation of azides into recombinant proteins for chemoselective modification by the Staudingerligation, PNAS 99: 19-24, 2002.

[0167] Expression system

[0168] In order to obtain high-level expression of cloned polynucleotides, polynucleotides encoding target proteins or polypeptides are usually subcloned into expression vectors containing strong promoters to direct transcription, transcription / translation terminators, and (if protein-encoding nucleic acids) ribosome binding sites for translation initiation. Suitable bacterial promoters are known to those of ordinary skill in the art and are described, for example, in Sambrook et al. and Ausubel et al.

[0169] Bacterial expression systems for expressing the target protein of the present invention can be used for, but are not limited to, Escherichia coli, Bacillus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, and Salmonella (Palva et al., Gene 22:229-235, 1983; Mosbach et al., Nature 302:543-545, 1983). Kits for such expression systems are commercially available.

[0170] The recombinant host cells of the present invention provide the ability to synthesize proteins comprising large useful amounts of unnatural amino acids. In one aspect, the composition optionally comprises, but is not limited to, at least 2 micrograms, at least 5 micrograms, at least 10 micrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 milligrams, at least 500 milligrams, at least 1 milligram, at least 10 milligrams, at least 100 milligrams, at least 1 gram or more of a protein comprising an unnatural amino acid, or an amount achievable using an in vivo protein production method (details on recombinant protein production and purification are provided herein). In another aspect, the protein is optionally present in the composition at a concentration including but not limited to at least 2 micrograms of protein per liter, 5 micrograms of protein per liter, 10 micrograms of protein per liter, at least 50 micrograms of protein per liter, at least 75 micrograms of protein per liter, at least 100 micrograms of protein per liter, at least 200 micrograms of protein per liter, at least 250 micrograms of protein per liter, at least 500 micrograms of protein per liter, at least 1 milligram of protein per liter, or at least 10 milligrams of protein per liter or more, in the form of, but not limited to, a cell lysate, a buffer, a pharmaceutical buffer, or other liquid suspension (including but not limited to, in a volume including but not limited to from about 1 nl to about 100 L or more).

[0171] The nucleotide sequence encoding the target protein may or may not contain a sequence encoding a signal peptide. When the polypeptide is to be secreted from the cell expressing it, a signal peptide is present. Such a signal peptide can be any sequence. The signal peptide can be prokaryotic or eukaryotic. (Coloma, J. Imm. Methods, 152, 89-104, 1992) describes a signal peptide for use in mammalian cells (mouse Ig κ light chain signal peptide). Other signal peptides include, but are not limited to, the -factor signal peptide from Saccharomyces cerevisiae (U.S. Pat. No. 4,870,008, which is incorporated herein by reference), the signal peptide of mouse salivary amylase (Hagenbuchle et al., Nature 289, 643-646, 1981), the modified carboxypeptidase signal peptide (Valls et al., Cell 48, 887-897, 1987), the yeast BAR1 signal peptide (WO 87 / 02670, which is incorporated herein by reference), and the yeast aspartic proteinase 3 (YAP3) signal peptide (see Egel-Mitani et al., Yeast 6, 127-137, 1990).

[0172] Bacterial expression techniques are known to those of ordinary skill in the art. Various vectors can be used for bacterial hosts. Vectors can be single copy or low or high multi-copy vectors. Vectors can be used for cloning and / or expression. In view of the large amount of literature on vectors, the commercial availability of many vectors and even the manuals describing vectors and their restriction maps and characteristics do not need to be extensively discussed here. As is well known, vectors generally involve markers that allow selection, which can provide cytotoxic agent resistance, prototrophy or immunity. Generally, there are multiple markers that provide different characteristics.

[0173] Bacterial promoters are any DNA sequences that can bind to bacterial RNA polymerase and initiate downstream (3') transcription of a coding sequence (e.g., a structural gene) into mRNA. The promoter will have a transcription initiation region, which is usually located near the 5' end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. Bacterial promoters may also have a second domain called an operon, which may overlap with an adjacent RNA polymerase binding site where RNA synthesis begins. Operators allow negatively regulated (inducible) transcription because gene repressors can bind to the operon and thereby inhibit transcription of specific genes. Constitutive expression can occur in the absence of negative regulatory elements (such as operons). In addition, positive regulation can be achieved by gene activator protein binding sequences, which, if present, are usually at the proximal end (5') of the RNA polymerase binding sequence. An example of a gene activator protein is a catabolite activator protein (CAP), which can help initiate transcription of the lac operon in Escherichia coli (E. coli) (Raibaud et al., Annu. Rev. Genet, 18: 173, 1984). Thus, regulated expression can be positive or negative, thereby enhancing or decreasing transcription.

[0174] The term "bacterial host" or "bacterial host cell" refers to a bacterium that can be or has been used as a recipient for recombinant vectors or other transfer DNA. The term includes the progeny of the original bacterial host cell that has been transfected. It should be understood that the progeny of a single parent cell are not necessarily identical to the original parent in morphology or genome or complete DNA complement due to accidental or deliberate mutations. The progeny expected by this definition include progeny of the parent cell that is sufficiently similar to the parent to be characterized by relevant characteristics (such as the presence of a nucleotide sequence encoding a target protein of the present invention).

[0175] The selection of suitable host bacteria for expressing proteins of the present invention is known to those of ordinary skill in the art. When selecting a bacterial host for expression, suitable hosts may include those showing good inclusion body forming ability, low proteolytic activity and overall robustness. Bacterial hosts are generally available from a variety of sources, including but not limited to the Bacterial Genetic Stock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA);And American Type Culture Collection ("ATCC") (Manassas, VA). Industrial / pharmaceutical fermentation generally uses bacteria derived from K strains (e.g., W3110) or bacteria derived from B strains (e.g., BL21). These strains are particularly useful because their growth parameters are well-known and robust. In addition, these strains are non-pathogenic, which is commercially important for safety and environmental reasons. Other examples of suitable E. coli hosts include but are not limited to BL21 strains, DH10B strains or derivatives thereof. In another embodiment of the inventive method, the E. coli host is an attenuated strain of protease, including but not limited to OMP- and LON-. The host cell strain can be a species of Pseudomonas, including but not limited to Pseudomonas fluorescens, Pseudomonas aeruginosa and Pseudomonas putida. Pseudomonas fluorescens biovar 1 (named MB101) is known to be useful for recombinant production and can be used for therapeutic protein production processes. Examples of Pseudomonas expression systems include systems available from Dow Chemical Company as host strains (Midland, MI, available on the World Wide Web dow.com).

[0176] Once a recombinant host cell strain is established (i.e., an expression construct has been introduced into a host cell and a host cell with an appropriate expression construct has been isolated), the recombinant host cell strain is cultured under conditions suitable for the production of the target protein or polypeptide. It will be apparent to those skilled in the art that the culture method of the recombinant host cell strain will depend on the nature of the expression construct used and the identity of the host cell. Recombinant host strains are typically cultured using methods known to those of ordinary skill in the art. Recombinant host cells are typically cultured in a liquid culture medium containing assimilable carbon, nitrogen, and inorganic salt sources, and optionally containing vitamins, amino acids, growth factors, and other protein culture supplements known to those of ordinary skill in the art. The liquid culture medium used to culture the host cells may optionally contain an antibiotic or antifungal agent to prevent the growth of undesirable microorganisms and / or compounds, including but not limited to antibiotics for selecting host cells containing the expression vector.

[0177] Recombinant host cells can be cultured in batch or continuous format, with cell harvesting or harvesting of culture supernatant being performed in batch or continuous format. For production in prokaryotic host cells, batch culture and cell harvesting are preferred.

[0178] After expression in a recombinant system, the target protein of the present invention can be purified. The target protein can be purified from a host cell or culture medium by various methods known in the art. The target protein of the present invention produced in a bacterial host cell may be poorly soluble or insoluble (in the form of inclusion bodies). In one embodiment of the present invention, amino acid substitutions can be easily made in a protein or polypeptide selected for the purpose of improving the solubility of a recombinantly produced protein using methods disclosed herein and methods known in the art. In the case of insoluble proteins, proteins can be collected from host cell lysates by centrifugation, and the cells can then be further homogenized. In the case of poorly soluble proteins, compounds including but not limited to polyethyleneimine (PEI) can be added to induce precipitation of partially soluble proteins. The precipitated protein can then be conveniently collected by centrifugation. Using various methods known to those of ordinary skill in the art, recombinant host cells can be destroyed or homogenized to release inclusion bodies from the cells. The destruction or homogenization of host cells can be carried out using well-known techniques, including but not limited to enzymatic cell destruction, ultrasonic treatment, dounce homogenization, or high pressure release destruction.

[0179] Then any of many suitable solubilizing agents known in the art can be used to dissolve insoluble or precipitated proteins or polypeptides. The protein or polypeptide can be dissolved with urea or guanidine hydrochloride. The volume of the dissolved protein or polypeptide should be minimized so that large batches of conveniently manageable batch production can be used. This factor may be important in a large-scale commercial environment, where recombinant hosts can be grown in batches of tens of thousands of liters of volume. In addition, when manufacturing proteins or polypeptides in a large-scale commercial environment, particularly for human pharmaceutical purposes, if possible, it is necessary to avoid using irritating chemicals or the protein product itself that can damage machines and containers.

[0180] In some cases, soluble proteins can be secreted into the periplasmic space or culture medium. In addition, soluble proteins can be present in the cytoplasm of the host cell. It may be necessary to concentrate the soluble proteins before performing the purification step. Standard techniques known to those of ordinary skill in the art can be used to concentrate soluble proteins, such as cell lysates or culture medium. In addition, standard techniques known to those of ordinary skill in the art can be used to destroy host cells and release soluble proteins from the cytoplasm or periplasmic space of the host cell.

[0181] Methods and techniques

[0182] The present disclosure encompasses methods and conventional techniques well known in the art. These include conventional methods of molecular biology, recombinant DNA techniques, biochemistry and cell biology, all of which are within the skill of the art. Such techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 3rd Edition (Sambrook et al., ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 2001); Oligonucleotide Synthesis: Methods And Applications (Methods in Molecular Biology), (Herdewijn, P., ed., Humana Press, Totowa, NJ); Oligonucleotide Synthesis (Gait, MJ, ed., 1984); Methods In Molecular Biology, (Humana Press, Totowa, NJ); Cell Biology: A Laboratory Notebook (Cellis, JE, ed., Academic Press, New York, NY, 1998); Animal Cell Culture (Freshney, RI, Ed., 1987); Introduction To Cell And Tissue Culture (Mather, JP and Roberts, PE, ed., Plenum Press, New New York, NY, 1998); Cell And Tissue Culture: Laboratory Procedures (Doyle, A. et al., ed., John Wiley and Sons, Hoboken, NJ, 1993-8); Gene Transfer Vectors For Mammalian Cells (Miller, JM et al., ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 1987); Current Protocols In Molecular Biology (Ausubel, FM et al., ed., Greene Pub. Associates, New York, NY, 1987); PCR: The Polymerase Chain Reaction, (Mullis, K.et al., editors, Birkhauser, Boston, MA, 1994); Short Protocols In Molecular Biology (John Wiley and Sons, Hoboken, NJ, 1999); Immunobiology 7 (Janeway, CA et al., Garland Science, London, UK, 2007); Antibodies (P. Finch, Stride Publications, Devoran, UK, 1997); Antibodies: A Practical Approach (D. Catty., editor, Oxford University Press, USA, New York, NY, 1989); Monoclonal Antibodies: A Practical Approach (Editor, Shepherd, P. et al., Oxford University Press, USA, New York NY, 2000); Using Antibodies: A Laboratory Manual (Harlow, E. et al., editor, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); The Antibodies (Zanetti, M. et al., eds., Harwood Academic Publishers, London, UK, 1995).

[0183] The present invention also relates to eukaryotic host cells, non-eukaryotic host cells and organisms for incorporating non-natural amino acids in vivo via orthogonal tRNA / RS pairs.Host cells are genetically engineered (including but not limited to conversion, transduction or transfection) using polynucleotides of the present invention or constructs comprising polynucleotides of the present invention (including but not limited to carriers of the present invention, which may be, for example, cloning vectors or expression vectors).

[0184] Several well-known methods for introducing target nucleic acids into cells are available, any of which can be used in the present invention. These include fusion of recipient cells with bacterial protoplasts containing the DNA, electroporation, projectile bombardment, and infection with viral vectors, etc. Bacterial cells can be used to amplify the number of plasmids containing the DNA constructs of the present invention. The bacteria are grown to the logarithmic phase, and the plasmids within the bacteria can be isolated by a variety of methods known in the art (see, e.g., Sambrook). In addition, kits for purifying plasmids from bacteria are commercially available (see, e.g., EasyPrep TM、FlexiPrep TM , both from Pharmacia Biotech; StrataClean from Stratagene TM ; and QIAprep from Qiagen TM ). The isolated and purified plasmid is then further manipulated to produce other plasmids for transfection of cells or incorporation into related vectors to infect organisms. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters that can be used to regulate the expression of a specific target nucleic acid. The vector optionally comprises a universal expression cassette containing at least one independent terminator sequence, sequences that allow the cassette to replicate in eukaryotic cells or prokaryotic cells or both (including but not limited to shuttle vectors), and selectable markers for prokaryotic and eukaryotic systems. The vector is suitable for replication and integration in prokaryotic cells, eukaryotic cells, or both. See, Gillam and Smith, Gene 8:81, 1979; Roberts, et al., Nature, 328:731, 1987; Schneider, E., et al., Protein Expr. Purif. 6(1):10-14, 1995; Ausubel, Sambrook, Berger (all supra). For example, ATCC provides a catalog of bacteria and bacteriophages that can be used for cloning, such as the ATCC Bacteria and Phage Catalog (1992) Gherna et al. (editor) published by ATCC. Other basic procedures and potential theoretical considerations for sequencing, cloning and other aspects of molecular biology have also been found in Watson et al. (1992) Recombinant DNA Second Edition Scientific American Books, NY. In addition, substantially any nucleic acid (and almost any labeled nucleic acid, whether standard nucleic acid or non-standard nucleic acid) can be customized or standard ordered from a variety of commercial sources such as Midland Certified Reagent Company (Midland, TX can be obtained on the World Wide Web at mcrc.com), The Great American Gene Company (Ramona, CA can be obtained on the World Wide Web at genco.com), ExpressGen Inc. (Chicago, IL can be obtained on the World Wide Web at expressgen.com), Operon Technologies Inc. (Alameda, CA) and many other sources.

[0185] Pill Box

[0186] Any composition described herein can be assembled together in a suitable container device in a medicine box. More particularly, all or part of the components used to design and construct or engineer the vector and / or plasmid of the present invention can be packaged together in a medicine box. One or more components of the present invention can be packaged separately or packaged together. In some embodiments, it is preferred that the vector and / or plasmid be packaged together with one or more components disclosed herein. In embodiments of the present invention, proteins, peptides, polypeptides (including antibody fragments) or nucleic acids encoding them can be packaged together, either as a single molecule or as a group of molecules. Alternatively, the components disclosed herein can be packaged and sold separately with instructions, which are in printed form or on a machine-readable medium, describing how to use them in combination with each other to design and construct vectors and / or plasmids, as disclosed herein.

[0187] In one embodiment, the medicine box may have a single container device, and / or it may have different container devices for other compounds. The components of the present invention can be provided in liquid form or dry powder form. In the case of providing the components with one or more liquid solutions, the liquid solution is an aqueous solution, particularly preferably a sterile aqueous solution. In other cases, the components of the medicine box can be provided in the form of dry powders. When a component (e.g., a reagent) is provided in the form of a dry powder, the powder can be reconstituted by adding a suitable solvent. It is conceivable that the solvent may also be provided in another container. The container device contemplated herein includes at least one vial, test tube, flask, bottle and / or other container device in a non-limiting manner.

[0188] Example

[0189] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes may be suggested to those skilled in the art and are to be included within the spirit and purview of the present application and the scope of the appended claims.

[0190] Example 1: Engineering of expression vector

[0191] The anti-CD3 Fab expression plasmids of the present invention were engineered in the E. coli NEB5α cloning strain (New England Biolabs, MA) as described below by a recombination-based cloning method using the Gibson Assembly kit (New England Biolabs, MA) or by using the QuikChange mutagenesis kit (Agilent Technologies, CA). Table 1 shows the three E. coli clones and production hosts used in this study, as well as their detailed genotypes.

[0192] Table 1. E. coli cloning and production host strains and genotypes

[0193]

[0194] Gibson Assembly Primers for amplifying various target genes (GOI) containing donor fragments had overlapping sequences of about 18-24 base pairs (bp) with the acceptor vector sequence for homologous recombination at their 5' ends and were synthesized at Integrated DNA Technologies ((IDT), San Diego, CA)). PCR fragments were amplified using a high-fidelity DNA polymerase mixture Pfu Ultra II Hotstart PCR Master Mix (Agilent Technologies, CA). PCR products were digested with Dpn1 restriction enzyme (NEB) for 2 hours at 37°C to remove plasmid background, then column purified using Qiagen PCR column purification kit (Qiagen) and quantified by Nanodrop (ThermoFisher). Acceptor vectors were linearized by digestion in the vector for 3 to 5 hours at the temperature recommended by the supplier with a unique restriction enzyme (NEB, MA), PCR column purified and quantified. The donor insert and the appropriately prepared acceptor vector were mixed at a molar ratio of 3:1, incubated at 50°C for 15 minutes, and then used for transformation into E. coli NEB5α strain (NEB) using the Gibson Assembly kit (NEB).

[0195] Recombinant was recovered by plating the Gibson Assembly mixture onto LB agar plates containing appropriate antibiotics. The next day, 4 to 6 fully separated single colonies were inoculated into 5mL LB+50μg / mL kanamycin sulfate (Sigma) or 100μg / mL carbenicillin (Teknova) culture medium, and grown overnight at 37°C. Qiagen plasmid DNA mini preparation kit (Qiagen) was used to separate the recombinant plasmid, and verified by DNA sequencing (Eton Biosciences, CA). Complete GOI region and 100bp upstream and 100bp downstream sequences were verified by using gene-specific sequencing primers.

[0196] QuikChange Mutagenesis (QCM):The amber variant containing TAG stop codon was created by using QuikChange Lightning site-directed mutagenesis kit (Agilent Technologies). QCM oligonucleotides were designed using QuikChange WebPortal (Agilent Technologies Inc.) and ordered from IDT (San Diego, CA). QCM PCR mixture contained 5 μl of 10x buffer, 2.5 μl of dNTP mixture, 1 μl (100ng) of plasmid template, 1 μl of oligonucleotide mixture (each 10uM concentration), 1 μl of QuikChange Lightning enzyme, 2.5 μl of Quick solution and 37 μl of distilled water (DW). DNA was amplified using the PCR program recommended by the kit, and only 18 cycles were performed.

[0197] After the PCR reaction was completed, the mixture was digested with the Dpn1 enzyme provided with the kit (Agilent Technologies) at 37°C for 2-3 hours and then run on a gel to check for the presence of the amplified PCR product. Then, 2.5 to 5 μl of the PCR product was transformed into the E. coli NEB5α strain. The recombinant plasmids of 4 to 6 colonies were then isolated and sequence verified as described above for Gibson Assembly.

[0198] Example 2: Engineering of expression strains

[0199] In order to prepare production strain of the present invention, chemical competent Escherichia coli W3110B60 host cells were transformed with sequence-verified plasmid DNA (50ng), recombinant cells were selected on 2xYT+1% glucose agar plates containing 50μg / mL kanamycin sulfate (Sigma) (or carbenicillin 100μg / mL, Teknova), and incubated overnight at 37°C. Then, by streaking three times in a row and incubating overnight at 37°C, single bacterium colonies from fresh transformation plates were bred three times on 2xYT+1% glucose agar plates containing 50μg / mL kanamycin sulfate or carbenicillin 100μg / mL. Finally, single bacterium colonies from the third streaking plate were inoculated into 20mL Super Broth (Fisher-OptiglowTM) containing 50μg / mL kanamycin sulfate (Sigma) (or carbenicillin 100μg / mL, Teknova), and incubated overnight at 37°C and 250rpm. The overnight grown culture was then diluted with glycerol to a final glycerol concentration of 20% (w / v).This cell suspension was then dispensed into 1 mL aliquots into several cryovials and frozen at -80°C as production strain vials.

[0200] After producing the glycerol bottle of production strain as mentioned above, the production strain was further verified by DNA sequencing and the phenotypic characterization of antibiotic resistance markers. In order to confirm that the production strain bottle has the correct plasmid in the production host, the plasmid was sequence verified. Twenty (20) mL LB containing 50 μg / mL kanamycin sulfate (or 100 μg / mL carbenicillin) was inoculated with the puncture from the cloned glycerol bottle, and grown overnight at 37 ° C, 250 rpm. Qiagen Miniprep test kit was used to separate plasmid DNA, and the presence of complete GOIORF in the separated plasmid was confirmed by DNA sequencing (Eton Biosciences, CA).

[0201] To further verify the strain genotype of each production strain, cells from the same vial were streaked onto four separate LB plates: LB containing 50ug / mL kanamycin sulfate, LB containing 15ug / mL tetracycline, LB containing 34ug / mL chloramphenicol, and LB containing 75ug / mL trimethoprim. All plates were then checked for positive growth, as expected given the strain genotype of the W3110B60 production host strain disclosed in Table 1.

[0202] Example 3: Fermentation process

[0203] The fermentation process for the production of anti-CD3 Fab-pAcF consists of two stages: (i) inoculum preparation and (ii) fermentor production. The inoculum was started from a single glycerol vial, which was thawed, diluted 1:1000 (v / v) into 50 mL of defined seed medium in a 250 mL baffled Erlenmeyer flask, and incubated at 37°C and 250 rpm. Prior to use, the fermentor was cleaned and autoclaved. The specified amount of basal medium was added to the fermentor and steam sterilized. Prior to inoculation, the specified amounts of kanamycin sulfate solution, feed medium, and P2000 antifoam were added to the basal medium. All solutions added to the fermentor after autoclaving were either 0.2 μm filtered or autoclaved prior to aseptic addition. The production fermentor was incubated at a target OD of 0.0004 by aseptically transferring the contents of the inoculum. 600 After inoculation, the culture was sampled at appropriate intervals to determine the OD 600 Temperature, pH and dissolved oxygen were monitored and controlled at specified set points of 37°C, 7.0 and >30%, respectively. pH was controlled by adding ammonium hydroxide solution or sulfuric acid. Dissolved oxygen was controlled by varying the agitation speed and increasing the oxygen composition of the sparged air / oxygen mixture. Antifoam was added during fermentation to control foaming.

[0204] When the cell density reaches OD 600>25, add a bolus of feed medium. When the cell density reaches OD 600 >50, feed medium was added at a constant flow rate of 0.094 mL / L starting volume / min for 32 hours until it dropped to 0.052 mL / L starting volume / min until the end of fermentation. Immediately after the start of feeding, the specified amount of pAcF solution was added aseptically to allow pAcF to be incorporated into the protein amino acid sequence. At the same time, the temperature was changed from 37°C used during growth to 27°C required for production. Production is controlled by the phoA promoter and begins when phosphate levels in the culture medium are depleted. Harvesting begins approximately 24 and 48 hours after induction of short-term and long-term fermentation processes, respectively.

[0205] Example 4: Plasmid retention assay

[0206] To measure plasmid retention of fermentation samples, glycerol (20% v / v) was added to 1 mL of fermentation broth at harvest and stored at -80°C for future analysis. 100 μL of this sample was removed and washed with water for 10 min. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 and 10 -8 Serially dilute the dilution into 900 μL LB broth. -4 , 10 -5 , 10 -6 , 10 -7 and 10 -8 The dilutions were plated on 2xYT+1% glucose agar plates. The plates were incubated overnight at 30°C. The next day, 100 colonies were picked from the dilution plates with fully separated colonies and replicates were plated on 2xYT+1% glucose agar and 2xYT+1% glucose+kanamycin (50 μg / mL) plates. The plates were incubated overnight at 30°C. After overnight incubation, colonies were counted and compared between 2xYT+1% glucose and 2xYT+1% glucose+kanamycin (50 μg / mL) plates. Plasmid retention was reported as the percentage of colonies grown on 2xYT+1% glucose plates compared to 2xYT+1% glucose+kanamycin (50 μg / mL) plates.

[0207] Example 5: Product titer quantification

[0208] To quantify product titer, 1 mL of fermentation broth was centrifuged (18,000 x g, 5 minutes) at various time points throughout the fermentation process. The supernatant was transferred to a separate 1.5 mL centrifuge tube, and the broth pellet and supernatant were retained and stored at -80°C for future analysis. As described below, the cell pellet was dissolved and titer was determined using ELISA or CEX assays.

[0209] Sample preparation: 1.0mL of E. coli cell mass from fermentation sample (cell paste) as described above is dried and weighed to determine sample quality. Lysonase bioprocessing reagent (Novagen) and Benzonase nuclease reagent (Novagen) are used to chemically lyse cell paste, and each is diluted 1:500 in BugBuster protein extraction reagent (Novagen). 1.0mL of Bugbuster / Lysonase / Benzonase mixture is added to 1.0mL of dry cell paste, and the resulting mixture is vortexed vigorously, then placed on a Thermos oscillator at 22°C for 20 minutes, while vibrating at 1000 revolutions per minute (rpm). After incubation is complete, the cell lysate is centrifuged at 18,000xg for 3 minutes to precipitate cell debris.

[0210] ELISA assay: ELISA, enzyme-linked immunosorbent assay, analyzes samples by immunoenzymatic assay to quantitatively measure the presence of anti-CD3 Fab. The above samples are then diluted in casein buffer (ThermoFisher) and then subjected to ELISA analysis. MSD plates (MSD) are coated with goat anti-human IgG Fd (Southern Biotech) specific for the heavy chain portion of anti-CD3 Fab. The coated ELISA plates are then washed with 1x KPL buffer (KPL) and blocked with casein blocking buffer. The samples containing anti-CD3 Fab and purified standards are then incubated on the plate with shaking at room temperature for 2 hours. The plate is washed with 1x KPL buffer, and an additional binding step with goat anti-human κ biotinylated (Southern Biotech) antibodies specific for the light chain portion of anti-CD3 Fab is performed to ensure that only complete Fab is detected. Sulfo-labeled streptavidin (MSD) is used for identification and quantification of captured anti-CD3 Fab proteins. 4x MSD read buffer (MSD) is diluted to 1x and then added to each well. The amount of anti-CD3 Fab in the samples was determined by extrapolation relative to the standard curve.

[0211] CEX assay:Then 200 μ L aliquots of the cell lysate supernatant as described above were filtered at 18,000 x g for 3 minutes through a 0.22 μ m PVDF centrifugal filter (Millipore). The filtered product was then analyzed by cation exchange chromatography (CEX) to determine the amount of anti-CD3 Fab present in the fermentation sample. Anti-CD3 Fab was separated from host cell protein contaminants using ProPac WCX-10, 4 x 100 mm CEX columns (ThermoFisher). Mobile phase A was used to capture anti-CD3 Fab and was composed of 20 mM sodium phosphate (pH 6.5), while mobile phase B, which was used to elute anti-CD3 Fab, was composed of 20 mM sodium phosphate, 200 mM sodium chloride (pH 6.5). The amount of anti-CD3 Fab in the sample was determined by interpolation of the peak area observed for the standard curve.

[0212] Example 6: Product quality and potency recovery

[0213] The anti-CD3 Fab of the present invention is each produced in E. coli cells and recovered from the whole cell lysate (WCL) supernatant. Cell lysis is performed at 4°C. The cells are dissolved in 100mM acetic acid, 100mM NaCl, 1mM EDTA (pH 3.5) in a volume equal to the original fermentation volume, thereby producing a post-lysis pH of 4.1-4.2. After lysis, the WCL is centrifuged at 15,900xg for 30 minutes at 4°C and filtered (0.8 / 0.2 microns) to remove precipitated proteins and cell debris. Capto S cation exchange chromatography is used to capture anti-CD3 Fab from E. coli WCL supernatant. RP-HPLC analysis is performed on a Capto S pool to determine the purity of the intact Fab versus the additionally trimmed and truncated heavy and light chain species co-purified with the intact Fab on the Capto S column. The recoverable titer was also estimated by calculating the total amount of protein recovered from the Capto S capture column and multiplying it by the percentage of intact Fab in the Capto S elution pool. When the harvested cell pellet was resuspended back into the fermentation harvest volume for solubilization and purification, the amount of intact Fab was divided by the initial Capto S load volume to estimate the fermentation titer.

[0214] Mass spectrometry: 5–10 μg of anti-CD3 Fab sample was loaded onto a reverse phase PLRP-S 2.1 mm x 150 mm, 8 μm, 4000 angstroms (A) analytical HPLC column (Agilent) for analysis. Purified samples and fraction collection samples were injected neat onto the analytical column. Mobile phase A consisted of 0.05% LC / MS grade TFA (FisherSci) in LC / MS grade HPLC water (FisherSci), while mobile phase B consisted of 0.05% LC / MS grade TFA in LC / MS grade acetonitrile (FisherSci). An Agilent 1200 series HPLC system with a binary pump coupled to an Agilent 6510ESIQ-Toff--LC / MS system was used for mass spectrometry analysis. Agilent Mass Hunter analysis software was used to qualitatively identify the mass of the chromatographically separated peaks. Mass spectrometry protein analysis software GPMAW was used to determine the theoretical mass of the primary sequence of the anti-CD3 Fab protein. The theoretical masses were compared to the measured masses to determine the identity of each peak.

[0215] Example 7: Anti-CD3 Fab expression system in E. coli

[0216] Figure 1 A schematic diagram of the anti-CD3 Fab expression cassette is shown, indicating the location of the UAA incorporation site within the heavy chain constant domain (CH1). This bicistronic operon is driven by the E. coli alkaline phosphatase (phoA) promoter that is induced when phosphate in the culture medium is depleted. The amino acid sequence of the anti-CD3 Fab consists of the vH and vL (variable regions of the immunoglobulin heavy chain and light chain, respectively) coding sequences plus the human γ1 (CH1) and κ (CL) constant regions. A secretory signal peptide (STII) is attached to both the heavy and light chains, directing the Fab to be secreted into the oxidized periplasm for proper folding and disulfide bond formation. The amber stop codon (TAG) is placed at the lysine (K) residue 129 (hereinafter referred to as HK129amber or HK129am) in the heavy chain constant domain (CH1) position 129 in the anti-CD3 Fab (shown in Figure 1 ). This TAG stop codon was encoded by the evolved Methanococcus jannaschii pAcF tyrRS / tRNA machinery at this site in pAcF (see, e.g., Wang L. et al., Science, 292, 498, 2001; Zhang Z. et al., Biochemistry, 42, 6735, 2003).

[0217] General scheme of expression vectors and corresponding production strain construction: Figure 2The general scheme of expression vector and its corresponding production strain construction are shown. Table 2 describes all expression vectors and their corresponding strains used in this study. In brief, as described above, expression vectors were initially constructed in Escherichia coli NEB5α (New England Biolabs, MA) cloning strains by Gibson Assembly method or QuikChange mutagenesis scheme. The sequence-verified expression vector DNA was then transformed into platform production host W3110 or W3110B60 strains (Table 1) to obtain corresponding production strains as described herein (Table 2).

[0218] Table 2. Production strains and corresponding expression plasmids

[0219]

[0220]

[0221] Example 8: Engineered 2797 strain with plasmid p56

[0222] Figure 3 A diagram of the expression plasmid p56 is shown. In designing and engineering this plasmid, an orthogonal system with an E9 RS synthetase and a tRNA cluster contained in the plasmid was utilized (see, e.g., U.S. Patent Publication Nos.: 2003010885, 20050009049, 20050208536, 20060233744). Overexpression of the E. coli proline synthetase ProS protein into a proprietary plasmid was also used in the design and engineering of the plasmid of the present invention to prevent proline misincorporation and to compensate for temperature-sensitive proS mutations within the chromosome of the proprietary platform production host W3110B60 strain to ensure plasmid maintenance (described, e.g., in Javahishvili, T. et al., ACS Chem. Biol., 9, 87, 2014).

[0223] A synthetic gene of the humanized anti-CD3 Fab sequence was engineered with an amber TAG stop codon at heavy chain amino acid HK129 flanked by SnaBI and SpeI restriction enzyme sites ( Figure 1 ). The engineered Fab gene was cloned into a proprietary E. coli expression vector using the Gibson Assembly kit (New England Biolabs, MA). After sequence verification, the plasmid was transformed into the E. coli production host W3110B60 strain, and the isolated single colonies were purified to make glycerol vials. These glycerol vials served as 2797 production clones for E. coli fermentation of Fab molecules. The DNA and amino acid sequences of the heavy and light chains of this Fab are shown in Table 3 below as SEQ.ID.No.: 1 to 11.

[0224] Table 3: Amino acid and DNA sequences of the anti-CD3 Fab-HK129am expression cassette

[0225]

[0226]

[0227]

[0228] The fermentation titer and plasmid retention of strain 2797 are shown in Figure 4 , 5A -B and Table 4 below.

[0229] Table 4. Plasmid retention of strain 2797

[0230]

[0231] Example 9: Engineered strains with alternatively partitioned locus B (parB) positioning and orientation

[0232] The production host cell line W3110B60 used to generate strain 2797 contains a mutation in the genomic proS proline synthase gene (W375R). This mutation renders the genomic ProS protein inactive above 34°C, creating a temperature-sensitive W3110B60 cell line with growth inhibited at 37°C. In addition to the anti-CD3 Fab gene of interest (GOI), the p56 plasmid in strain 2797 also contains the E. coli proS gene for expression of the wild-type E. coli ProS protein. This enables growth and provides selective pressure to maintain the GOI plasmid at 37°C. However, it may not provide an advantage for maintenance of the plasmid when production temperatures are below 34°C. The kanR gene for kanamycin resistance is also present in the p56 plasmid ( Figure 3 ) when the antibiotic kanamycin was added to the culture medium to provide selective pressure for plasmid maintenance. Even with these two components, poor plasmid retention was observed in 2797 cells collected at various time points during the fermentation (Table 4).

[0233] In order to ensure that the plasmid of robustness is maintained during the whole fermentation operation, no matter Fab production temperature or any other process limitation, various strategies are studied.One strategy is to ensure that the appropriate plasmid is separated during bacterial cell division so that each daughter cell inherits the same plasmid.It has been shown in the literature that in some bacterial plasmid separation systems, a specific gene called plasmid distribution locus (par) ensures this point by killing the daughter cells that have lost the plasmid during cell division (Ebersbach G. and Gerdes K., Annu. Rev. Genet. 39, 453, 2005). In addition, other findings in the literature also indicate that incorporation of a 580-bp minimal partitioning B parB (hok / sok) locus (SEQ.ID.NO:12) (originally from E. coli plasmid R1) on a plasmid can improve its maintenance and stability in many bacteria, including E. coli (Gerdes K., Nature Biotech. 6, 1402, 1988; Gerdes K. et al., Mol. Microbiol. 4, 1807, 1990; Gerdes K. and Neilsen Al, J. Mol. Biol. 226, 637, 1992).

[0234] Therefore, the inventors attempted to clone a synthetic 580-bp parB locus gene (DNA2.0, Menlo Park, CA) with a forward (F) transcriptional direction of its host killer (hok) gene (SEQ.ID.NO: 13) and amino acids (SEQ.ID.NO: 14) relative to the transcriptional direction of the downstream Fab gene between the AfeI and NotI restriction enzyme sites in plasmid p56. This strategy produced a synthetic engineered plasmid p96 with a 271bp deletion in the plasmid backbone just upstream of the phoA promoter region. All other coding regions (CDS) and their transcriptional directions (including Fab genes, kanR and proS genes) remained unchanged. Plasmid p96 was then transformed into the standard production host W3110B60 strain as described in the above examples to obtain production strain 2835.

[0235] Table 5: DNA and protein sequences of the parB locus, FkpA and Skp chaperones

[0236]

[0237]

[0238]

[0239] Production strains 2797 and 2835 were then compared in a high cell density fermentation process as described above. Figure 4The production titers of strains 2797 and 2835 under short-term and long-term fermentation conditions were compared. Table 6 shows the plasmid retention data for these two strains during two different fermentation processes. After the long fermentation process was optimized to retain plasmids, the typical plasmid retention of strain 2797 at 48 hours varied between 0% and 44%, with 1% plasmid retention measured at 54 hours. As expected and shown, incorporation of the parB locus in strain 2835 significantly improved plasmid retention. Two fermentation runs using strain 2835 showed 100% plasmid retention in both processes and at both time points (22 hours and 54 hours; Table 6).

[0240] Table 6. Plasmid retention of parB-containing strains and parent strain 2797

[0241]

[0242] Despite the improved plasmid maintenance, the use of the 2835 cell line resulted in approximately 40% reduction in cell pellet titer for both short-term and long-term fermentations ( Figure 4 ). This was unexpected, since theoretically, improved plasmid retention should at least maintain the same productivity, even if there was no improvement at all. An improvement in titer was later observed by insertion of the parB locus without deletion of plasmid backbone sequences, as described below.

[0243] Example 10: Engineering of ParB-containing vectors and strains without plasmid sequence deletion

[0244] As described above, as expected from the literature, insertion of the parB locus between the AfeI and NotI restriction enzyme sites in the forward (F) orientation resulted in improved plasmid retention (100%). However, unexpectedly, it also reduced the intact Fab titer in the cell mass by about 40%. In order to recover this production loss, four new expression plasmids (p117, p118, p119 and p120; Table 2) without any deletions in the plasmid backbone sequence were engineered. The unique restriction enzyme sites of AfeI and ZraI within the plasmid backbone were utilized. The same parB locus was cloned in two positions, either forward (F) or reverse (R), relative to the Fab CDS. This strategy produced four new production strains, 2884, 2885, 2886 and 2887, (Table 2), which were then compared with the original strain 2797 for plasmid retention and production titer in high cell density fermentations ( Figure 5A -B and Table 6).

[0245] The new production cell lines are 2884 (anti-CD3 Fab, parB-AfeI-F), 2885 (anti-CD3 Fab, parB-AfeI-R), 2886 (anti-CD3 Fab, parB-ZraI-F) and 2887 (anti-CD3 Fab, parB-ZraI-R), where AfeI or ZraI indicates the restriction enzyme site for cloning the parB locus, and F (forward) indicates that the transcription of the hok gene is in the same orientation as the Fab CDS, where R (reverse) indicates that the transcription of the hok gene is in the reverse orientation compared to the Fab CDS. These production cell lines were compared with 2797 (anti-CD3 Fab) without the parB locus.

[0246] As expected, all four new parB cell lines showed 100% plasmid retention 48 hours after induction (Table 6). This demonstrated a significant improvement over 2797 without the parB locus, where plasmid retention at the end of fermentation varied from as low as 0% to a maximum of 44% measured (depending on the fermentation process used) (see Table 6 and Examples above). Surprisingly, the location and orientation of the parB locus within the plasmid had a significant effect on anti-CD3 Fab titers. Three of the four new strains (strains 2885, 2886, and 2887) showed significantly improved cell mass relative to the 2797 control strain ( Figure 5A ) and culture medium ( Figure 5B ) titer. In addition to the above-described changes in the position and orientation of the parB locus from 2835, in strains 2884 to 2887, parB was cloned to avoid deleting any portion of the plasmid sequence. In a separate study, it was observed that deleting portions of the plasmid sequence upstream of the phoA promoter region had a negative impact on Fab titer (data not shown). The increase in Fab titer observed in most of these constructs may be supported by repositioning parB away from this region and retaining this sequence intact.

[0247] Only strain 2884 had lower cell mass and culture titer than control strain 2797 ( Figure 5A-5B ). The only difference between strains 2884 and 2835 described herein is the deletion of a 271-bp plasmid sequence between the AfeI and NotI restriction enzyme sites with the same parB-F orientation. Therefore, since the deletion of the plasmid sequence at this position or its absence resulted in lower Fab titers (strain 2835 compared to 2884), it was concluded that parB orientation is more important than positioning for increasing titer, given the improved titer of strain 2885 with a parB-R orientation.

[0248] To determine the best strain to optimize the expression process, further studies were performed using strains 2886 and 2887, in which parB is positioned further away from the phoA promoter region. Strain 2886 was selected because it had the highest titer, measuring 236 mg / L 32 hours after induction (n=average of 3 fermentations; data not shown). Figure 6 A map of plasmid p119 contained in strain 2886 is shown, along with the optimal location and orientation of the parB locus for cloning.

[0249] Example 11: Design and function of periplasmic chaperone proteins in a compatible two-plasmid expression system

[0250] To determine the involvement of molecular chaperones in optimizing or increasing protein titer in E. coli, two well-known bifunctional periplasmic chaperones with peptidylprolyl isomerase activity, FkpA and Skp, were analyzed.

[0251] To this end, two compatible plasmids with the p15A origin of replication (10-15 copies / cell) were constructed by cloning the FkpA or Skp gene. These kanamycin resistance (KanR) plasmids were co-transformed with an anti-CD3 Fab expression plasmid with ampicillin resistance (AmpR) and the pBR322 origin of replication (30-50 copies / cell) to test the effect of co-expression of the periplasmic chaperone ( Figure 7 A).

[0252] For this study, the wild-type W3110 expression host strain was used, given that the standard production host W3110B60 strain has a KanR marker inserted into its chromosome (Table 1). Figure 7 As shown in A, all three species-specific cell lines in this study were co-transformed with two plasmids. Plasmid 1 contained anti-CD3 Fab + ampR + parB (where parB was cloned with a 271-bp deletion between the AfeI and NotI restriction enzyme sites) in all three production strains. For the periplasmic chaperone strains 2840 and 2841, plasmid 2 contained the chaperones FkpA and Skp, respectively, behind an inducible tetracycline promoter (pTetA) with a kanamycin resistance (kanR) marker, where the fkpA and skp genes were induced by adding 50 parts per billion (ppb) tetracycline in the culture medium ( Figure 7 A). For control strain 2939, plasmid 2 contained the kanamycin resistance (kanR) marker but no chaperone proteins. The DNA and protein sequences of the FkpA and Skp chaperone proteins are included as SEQ ID NOs: 15 to 18 in Table 5.

[0253] Using the above examples and Figure 4All fermentations were performed using the short fermentation process shown. For all three strains, 50 μg / mL carbenicillin and 50 μg / mL kanamycin were added to the medium to maintain both plasmids. For strains 2840 and 2841 (Table 7), phosphate (PO4) was depleted by adding pAcF to 2 mL of 50 μg / mL tetracycline (50 ng / mL final concentration) to induce expression of the chaperone proteins. Cell pellet ELISA titers were Figure 8 Contrary to expectations, no increase in pellet titer was observed for co-expression of the periplasmic chaperones FkpA and Skp. There was no leakage of the culture medium for any of these strains (data not shown).

[0254] Table 7. Compatibility of production strains and corresponding expression plasmids in the two-plasmid system

[0255]

[0256] Note that the observed lack of improved Fab expression may be attributed to the expression of periplasmic chaperones not being coupled to their natural E. coli promoters, because they are artificially driven by the inducible tetracycline promoter (pTetA) disclosed herein. In E. coli, the expression of FkpA and Skp genes is driven by another sigma factor E (sigma E), which is involved in various periplasmic stress sensing, including misfolded / unfolded protein stress (Merdanovic M. et al., Annu. Rev. Microbiol. 2011, 65, 149). This allows, when necessary, that is, when cells sense misfolded / unfolded protein stress, to induce the expression of these chaperones. Based on this principle, the inventors attempted to determine the impact of the natural promoter system, as described below.

[0257] Example 12: Design and function of periplasmic chaperone proteins in a single plasmid expression system

[0258] As described above, given that no improvement in Fab titer was observed in the compatible two-plasmid system, a single-plasmid system was examined. In this system, the chaperone genes FkpA and Skp were cloned behind their native promoters into the same plasmid as the anti-CD3 Fab with parB-ZraI-F in the 2886 strain (see Figure 7 B).

[0259] The cell lines used in this study were 2886 (anti-CD3 Fab, parB-ZraI-F) as a control, 2909 (anti-CD3 Fab, parB-ZraI-F, fkpA) for FkpA co-expression, and 2910 (anti-CD3 Fab, parB-ZraI-F, skp) for Skp co-expression. Plasmids p134 and p135 (Table 2) contain the FkpA and Skp chaperones, respectively, with their native promoter sequences cloned in the forward (F) orientation relative to the parB locus and Fab orientation. This strategy thus couples expression to cell physiology (i.e., misfolded / unfolded protein stress) rather than to uncoupled tetracycline-inducible expression as in the two-plasmid system (see Fig.10 , p134 plasmid map).

[0260] Co-expression of the periplasmic chaperones FkpA or Skp in this manner resulted in total Fab production significantly exceeding 2886 titers ( Figures 9A-9B ). However, Skp co-expression resulted in a large infiltration of Fab into the culture medium, and the titer of 2910 cell mass was no higher than 2886 at 48 hours after induction. By co-expression of FkpA, most of the Fab was retained in the cell mass. A titer of 424 mg / L ELISA was measured for 2909, compared to 245 mg / L ELISA titer of 2886 measured on the same day. Therefore, it is clear that the co-expression of chaperone proteins is very important for the production of recombinant proteins. From these data, 2909 with FkpA chaperone proteins was selected for further development.

[0261] Example 13: Optimization of the FkpA chaperone position and orientation relative to the Fab and ParB loci within the plasmid

[0262] As disclosed above in strain 2909, in a single plasmid expression system, the FkpA chaperone protein was cloned in the forward orientation behind the parB locus ( Fig.10 When the same FkpA chaperone gene was expressed from a separate plasmid as in the dual plasmid expression system of strain 2840 (see Figure 7 AB and Figure 8 ) did not increase Fab titers, thus emphasizing the importance of aligning co-chaperone expression with cellular physiology.

[0263] As described in the above examples, based on the data obtained about the position and orientation effects of the parB locus in the expression plasmid relative to the Fab orientation, the inventors speculated that the position and orientation of the chaperone gene containing FkpA may also affect the Fab titer. In order to examine this aspect of the invention, the fkpA gene was designed and engineered by cloning the fkpA gene in front of or behind the parB locus in For and Rev orientations. This strategy allows the impact of chaperone gene co-expression on recombinant protein production to be evaluated at three different levels: 1) promoter effects, 2) position effects, and 3) orientation effects.

[0264] To this end, three new plasmids (p199, p200, and p201) were designed and constructed by changing the position and orientation of the FkpA chaperone relative to the parB-F position as shown in Table 8. Three (3) new production strains (strains 3004, 3005, and 3006) carrying these new plasmids were tested for their effect on Fab expression titers. Fig.11 As shown, the location and orientation of the FkpA chaperone relative to the parB-F orientation significantly affected Fab titer. No leakage of Fab into the culture medium was observed (data not shown).

[0265] Table 8. Effect of location and orientation of the parB locus and FkpA chaperone on Fab expression titers.

[0266]

[0267]

[0268] Example 14: Effect of host strain on anti-CD3 Fab titer and product quality

[0269] It is known in the literature that antibody Fab produced in E. coli is trimmed by proteases in the periplasm under production conditions without any unnatural amino acid insertion (Battersby, JE et al., Journal of Chromatography A, 927, 61, 2001). In order to study how the orthogonal amber inhibition system affects the Fab product quality, a Fab capture column was developed by cation exchange using Capto S resin, which captures the complete Fab as well as several additional fragments. The strain 2886 protein from plasmid p119 (Table 2) with a parB locus was used for this study.

[0270] Analytical characterization of the Capto S elution pool from strain 2886 by RP-HPLC plus mass spectrometry (MS) revealed that the two captured fragments were proteolytically trimmed HC and LC Fab fragments (Battersby, JE et al., Journal of Chromatography A, 927, 61, 2001), and an additional translationally truncated HC fragment due to the amber stop codon located between the HC ( Fig.12 AB). By A280 absorbance percentage area from RP-HPLC on Capto S pool, intact Fab was found to account for only about 20% of the captured protein. The remaining Fab fragments captured were light chain trimmed from the N-terminus (aa 1 to 110) (about 20%), heavy chain trimmed from the N-terminus (about 20%) (aa 1 to 217), and heavy chain truncated at the amber stop codon at the HK129 position for pAcF incorporation (about 40%) (aa 1 to 141) ( Fig.12 AB). The only fragment not captured in this Capto S column is the C-terminal light chain trimmed fragment (aa 111 to 217).

[0271] Example 15: Design, Engineering and Expression Analysis of Vectors with Various Heavy Chain Extensions

[0272] Construction of heavy chain (HC) C-terminal extension strains: As disclosed in the above examples, RP-HPLC and intact mass on the Capto S elution pool identified various Fab fragments that were present as a high percentage of the total Fab protein. The possible protease responsible for Fab cleavage is the periplasmic serine endoprotease Prc (for processing the C-terminus), also known as the tail-specific protease (Tsp) (Chen C. et al., Biotechnology and Bioengineering, 85, 463, 2004). It is believed that Prc recognizes and binds to the C-terminus of the protein, and then cleaves in loose or unfolded regions within the protein sequence (Keiler et al., Protein Science, 4, 1507, 1995). The site of cleavage is determined by the secondary or tertiary structure of the protein rather than the primary sequence, and therefore, Prc has a broad sequence specificity for proteolytic sites. However, it has been demonstrated that the C-terminal sequence of a protein affects its cleavage by Prc, and that modifying the C-terminal sequence of a protein can alter the amount of cleavage of the protein by Prc (Keiler KC and Sauer RT, The Journal of Biological Chemistry, 271, 2589, 1996).

[0273] In this study, the Fab heavy chain C-terminal sequence was modified with the goal of reducing Prc C-terminal binding and thereby reducing Prc cleavage. This strategy was pursued to improve the purity of the starting material for the Capto S capture column used for purification, as well as to increase intact Fab titer by reducing the percentage of HC generated as trimmed species, thereby increasing the amount of HC available for assembly into intact Fab. In the Fab design, the heavy chain sequence was based on KSC 220 Terminate, where C 220 Involves only interchain disulfide bond formation with light chains. 214 Unlike the terminating light chain (LC) sequence, the native HC sequence extends beyond the CH1 domain in the Fab fragment to the hinge region CH2 domain and finally terminates at the CH3 domain. Based on the criteria reported in the literature (Keiler KC and Sauer RT, The Journal of Biological Chemistry, 271, 2589, 1996), in order to determine whether the Prc recognition at the HC C-terminal sequence can be altered by extending the HC C-terminal sequence beyond the C220 residue, the C-terminal end was extended from the sequence D221 KTHT 225 Several heavy chain sequence variants were designed with 1 to 5 amino acids of the native sequence extended to the hinge region. A variant with T reported for Genentech's Lucentis Fab (ranibizumab) was also designed. 225 To L 225 Substituted variants (Table 2) (see the World Wide Web at drugbank.ca / drugs / DB01270). All cell lines used in this study had the parB locus and the FkpA chaperone protein was co-expressed from an anti-CD3 Fab plasmid as 2909 (anti-CD3 Fab, parB-ZraI-F, fkpA).

[0274] Testing the effect of HC C-terminal extension: A 50 mL sample was purified by passing it through a Capto S column and then through a Capto SA column. 280 The recoverable potency was estimated by multiplying the recovery by the RP-HPLC purity in the Capto S pool ( Fig.13A ). Fig. 13B RP-HPLC purity results for Capto S pools from 50 mL purification are shown in .

[0275] All five heavy chain C-terminal extension variants had higher recoverable titers when compared to the control 2909, reaching 610 mg / L for 2918 ( Fig.13AThese heavy chain variants also showed a higher percentage of intact Fab in the Capto S pool than the control 2909 (30% to 42% intact for the heavy chain variants vs. 25% intact for the control 2909, Fig. 13B Surprisingly, while the percentage of trimmed heavy chain species decreased slightly (as expected from reduced Prc proteolysis), a larger decrease in the percentage of truncated heavy chains was observed (31% to 38% truncated for heavy chain variants vs. 39% truncated for control 2909). This suggests that not all "truncated" HC species are necessarily a result of low inhibition efficiency and pAcF incorporation, and that some of these species may also be due to proteolysis. Alternatively, changes in the heavy chain C-terminal sequence may independently affect inhibition efficiency.

[0276] Although the heavy chain C-terminal extension variants did show higher titers and higher percentages of intact Fab, indicating reduced binding and proteolysis of Prc, there was no direct correlation between the percentage of intact Fab and Fab titer. Among the heavy chain extension variants, 2918 had the highest recoverable titer (610 mg / L), but the lowest percentage of intact Fab (30%). There are many variables that affect titer, and changing the protein sequence can, for example, affect expression, secretion to the periplasm, folding (including cis-trans proline isomerization and disulfide bond formation), as well as affecting the proteolysis of Prc.

[0277] If available Fig. 13B The heavy chain C-terminal sequence KSC 220 DKTHT 225 The 2917 strain had the lowest percentage of trimmed LC, trimmed HC fragments, and truncated HC fragments with the highest percentage of intact Fab titers. Although strain 2917 Fab titers were lower compared to the other variants, this strain was selected for further process development, cell line generation, and in vivo studies due to better overall protein quality.

[0278] It should be understood that the methods and compositions described herein are not limited to the specific methods, protocols, cell lines, constructs and reagents described herein, and may vary accordingly. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the scope of the methods and compositions described herein, which will only be limited by the appended claims.

[0279] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0280] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention disclosed and described herein belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention described herein, preferred methods, devices and materials are now described.

[0281] For the purpose of describing and disclosing, for example, constructs and methodologies described in the publications and that can be used in conjunction with the presently described invention, all publications and patents mentioned herein are incorporated herein by reference in their entirety. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. In this regard, nothing herein should be construed as an admission that the inventors described herein are not entitled to antecedent disclosures due to prior invention or for any other reason.

[0282] Although the foregoing invention has been described in detail for the purpose of clarity and understanding, it will be clear to those skilled in the art by reading this disclosure that various changes can be made in form and detail without departing from the true scope of the invention. For example, all techniques and equipment described herein can be used in various combinations. It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and it is suggested that those skilled in the art make various modifications or changes thereto, and they will be included within the spirit and authority of the present application and within the scope of the appended claims.

[0283] Incorporated by Reference

[0284] All publications, patents, patent applications and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application and / or other document was individually indicated to be incorporated by reference for all purposes.

Claims

1. A vector for optimizing protein expression, the vector comprising a nucleic acid sequence encoding a target protein and a nucleic acid sequence encoding one or more components, the target protein having a non-naturally encoded amino acid site specifically incorporated, wherein the one or more components are selected from SEQ.ID.NO.: 12, 15 and 17.

2. A vector as claimed in claim 1, wherein the one or more components are auxiliary proteins or factors or genetic elements.

3. A vector as claimed in claim 2, wherein the auxiliary protein or factor is a chaperone protein, or, wherein the genetic element is the partitioning B locus (parB).

4. The vector of claim 1, further comprising an antibody heavy chain C-terminal extension variant, optionally wherein the antibody heavy chain C-terminal extension variant comprises one or more amino acids.

5. The vector according to claim 1, wherein: a. parB is located between the Afe1 and Not1 sites of the nucleic acid sequence encoding the target protein in a forward transcription orientation; b. parB is located at the Afe1 or Zra1 site of the nucleic acid sequence encoding the target protein in a forward transcription orientation; or c. parB is located at the Afe1 or Zra1 site of the nucleic acid sequence encoding the target protein in a reverse transcription orientation.

6. The vector of claim 3, wherein the chaperone protein is Skp or FkpA, or, wherein the chaperone protein is Skp and FkpA.

7. The vector of claim 6, further comprising a partitioning B locus (parB).

8. The vector of claim 1, wherein the target protein is selected from a biotherapeutic agent, an immunogen, an antibody, an antibody fragment, or a variant thereof.

9. The vector of claim 8, wherein: a. The biotherapeutic agent is a vaccine; b. wherein the target protein is: i. a cytokine, chemokine, growth factor, growth factor receptor, interferon, interleukin, inflammatory molecule, oncogene product, peptide hormone, signal transduction molecule or steroid hormone receptor; or ii. HER2, CD-70, PSMA, 5T4, EGFR, TROP2, CD3, IL-2, IL-3, IL-10, IL-12, IL-15, IL-21, GPC3, DLL3, ROR1, leptin, growth hormone releasing peptide, FGF-1, FGF-19, FGF-21, FGF-23, HGH, FcR, insulin, IGF1, TNFR1, TRAIL, EPO and analogs, bispecific molecules or fragments thereof; c. The antibody fragment is: i. Fab, Fab', F(ab')2, Fv fragment, single-chain antibody fragment (scFv), disulfide-stabilized scFv (dsFv)), diabody (Db), BiTE (bispecific T cell engager), DART (dual affinity retargeting) or tandem diabody (TandAb); ii. Fab; or iii. anti-CD3 Fab; or d. The antibody is a full-length antibody, optionally, wherein the full-length antibody is an IgG1, IgG2, IgG3 or IgG4 antibody.

10. A recombinant cell comprising a nucleic acid sequence encoding a target protein and a nucleic acid sequence encoding one or more components, wherein the target protein has a non-naturally encoded amino acid site specifically incorporated, wherein the one or more components are selected from SEQ.ID.NO.: 12, 15 and 17.

11. The recombinant cell of claim 10, wherein the cell comprises the chaperone protein Skp and the partitioning B locus (parB), or, wherein the cell comprises the chaperone protein FkpA and the partitioning B locus (parB).

12. The recombinant cell of claim 10, wherein the cell comprises an antibody heavy chain C-terminal extension variant.

13. The expression plasmid of any one of claims 1-12, wherein the plasmid is p56, p94, p96, p117, p118, p119, p120, p134, p135, p141, p142, p143, p144, p145, p199, p200 or p201.

14. A method for producing a target recombinant protein incorporating non-naturally encoded amino acids, the method comprising expressing the protein in a bacterial cell and introducing a nucleic acid sequence encoding one or more components, wherein the one or more components are selected from SEQ.ID.NO.: 12, 15 and 17.

15. The method of claim 14, wherein the method is used to increase the yield of a protein having a specifically incorporated non-natural amino acid site, optionally wherein the non-natural amino acid is O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, p-propargyloxy-L-phenylalanine, tri-O-acetyl-GlcNAcβ-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L -phenylalanine, L-phosphoserine, phosphonoserine, phosphotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, p-acetylphenylalanine, p-nitrophenylalanine, p-sulfotyrosine, p-carboxyphenylalanine, o-nitrophenylalanine, m-nitrophenylalanine, p-borylphenylalanine, o-borylphenylalanine, m-borylphenylalanine, p-aminophenylalanine, o-aminophenylalanine, m-aminophenylalanine, p-acylphenylalanine, o-acylphenylalanine, m-acylphenylalanine, p-OMephenylalanine, o-OMephenylalanine, m- OMe phenylalanine, p-sulfophenylalanine, o-sulfophenylalanine, m-sulfophenylalanine, 5-nitroHis, 3-nitroTyr, 2-nitroTyr, nitro-substituted Leu, nitro-substituted His, nitro-substituted De, nitro-substituted Trp, 2-nitroTrp, 4-nitroTrp, 5-nitroTrp, 6-nitroTrp, 7-nitroTrp, 3-aminotyrosine, 2-aminotyrosine, o-sulfotyrosine, 2-sulfooxyphenylalanine, 3-sulfooxyphenylalanine, o-carboxyphenylalanine, m-carboxyphenylalanine, p-acetyl-L-phenylalanine, p-propargyl-phenylalanine acid, o-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine or p-propargyloxy-phenylalanine, Preferably, the unnatural amino acid is p-acetylphenylalanine.

16. An isolated cell or cell line according to claim 14.

17. The isolated cell or cell line of claim 16: a. wherein in the presence of one or more chaperone proteins and a parB locus, the yield of the protein containing the unnatural amino acid is at least 0.25 times or more; b. wherein the parB locus is located at the Afe1 site of the nucleic acid sequence encoding the target protein in a reverse orientation; or c. The isolated cells or cell lines are used to optimize plasmid retention in production cells or cell lines.

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