Recombinant production of authentic human proteins using human cell expression systems

Abstract

The present invention relates to novel expression cassettes and vectors for efficiently producing authentic recombinant human proteins from stable cultures of novel human cell lines, the authentic recombinant proteins produced therefrom, and antibodies raised against those authentic recombinant proteins.

1 . An expression vector, comprising a cassette which comprises the following operably linked expression elements:
 (A) a cytomegalovirus enhancer element sequence;   (B) a human promoter sequence selected from the group consisting of (i) a human actin promoter sequence, (ii) a human serum albumin promoter sequence, and (iii) a human fibrinogen promoter sequence;   (C) a human globin gene intron sequence; and   (D) a human signal peptide sequence.   
     
     
         2 . The expression vector of  claim 1 , further comprising a desired polynucleotide positioned downstream of the signal peptide sequence and operably linked to elements (A), (B), (C), and (D) of  claim 1 . 
     
     
         3 . The expression vector of  claim 2 , wherein either (i) the desired polynucleotide is operably linked to a termination signal sequence that is located in the expression vector downstream of the human signal peptide sequence, or (ii) the desired polynucleotide itself comprises a termination signal sequence. 
     
     
         4 . The expression vector of  claim 1 , wherein the human promoter sequence is a functional human beta-actin promoter sequence. 
     
     
         5 . The expression vector of  claim 1 , wherein the CMV enhancer element is the CMV immediate early enhancer sequence. 
     
     
         6 . The expression vector of  claim 1 , wherein the signal peptide sequence is an immunoglobulin superfamily 8 signal peptide, or an alpha-fibrinogen signal peptide. 
     
     
         7 . The expression vector of  claim 2 , wherein the desired polynucleotide is operably linked to a CMV immediate early enhancer, a beta-actin promoter sequence, a human globin gene intron sequence, and an immunoglobulin superfamily 8 signal peptide. 
     
     
         8 . The expression vector of  claim 2 , wherein the desired polynucleotide is operably linked to a CMV immediate early enhancer, a beta-actin promoter sequence, a human globin gene intron sequence, and an alpha-fibrinogen signal peptide. 
     
     
         9 . The expression vector of  claim 2 , wherein the desired polynucleotide encodes a cytokine. 
     
     
         10 . The expression vector of  claim 9 , wherein the encoded cytokine is selected from the group consisting of Activin A, Activin B, Activin A/2xINHbA, Activin B/2xINHbB, AMH/MIS, Artemin, BDNF, BMP2, BMP15/GDF9B, BMP2/BMP2A, BMP3/Osteogenin, BMP4, BMP4/BMP2B, BMP5, BMP7/OP-1, BMP1, BMP10, BMP15/GDF9B, β-NGF, Cystatin C, Delta 1, EGF, Erythropoietin (EPO), FGF acidic, FGF basic, FGF10, FGF5, FGF7, FGF8b, FLT3 ligand, G-CSF, GDF15, GDF2/BMP9, GDF3, GDF5/BMP14, GDF8/myostatin, GDF9, GDNF, GM-CSF, HGF, HGH, IFN-α2A, IFN-α2B, IFN-γ, IFN-β1, IGF I, IGF II, IGF IIv1, IGF IIv2, IL10, IL11, IL12, IL15, IL17/IL17A, IL17F, IL1β, IL2, IL23, IL27, IL28A/IFN-lambda-2, IL28B/IFN-lambda-3, IL29/IFN-lambda-1, IL1B, IL2 IL3, IL32, IL35, IL4, IL5, IL6, IL7, IL8, IL9, Inhibin A/INHa&INHbA, Inhibin B/INHa&INHbB, Inhibin C/INHa&INHbC, Inhibin E/INHa&INHbE, LEFTYB, LEFTY1/LeftyB, M-CSF, mouse CSF, mouse SCF, NODAL, Noggin, NT3 (neurotrophin3), Oncostatin M, PDGFα, PDGFβ, Persephin, SCF, SDF1α, SHH, Somatotropin, TGF β1, TGF β2, TGF β3, TGFβ4/LEFTY2/LeftyA, TNF α, TPOα, VEGF121aa, VEGF165aa, WIF1, WNT1, Wnt10A, Wnt10B/12, Wnt11, Wnt16, Wnt2, Wnt2B/13, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8B, and Wnt9A/14. 
     
     
         11 - 65 . (canceled)