Methods and Materials for Increasing Expression of Recombinant Polypeptides

Abstract

The present invention provides novel methods and materials for increasing the expression of recombinant polypeptides. Methods and materials of the invention allow increased expression of transcription units that include recombinant DNA sequences which encode polypeptides of interest. The present invention provides expression vectors which contain multiple copies of a transcription unit encoding a polypeptide of interest separated by at least one selective marker gene and methods for sequentially transforming or transfecting host cells with expression vectors to increase transcription unit dosage and expression.

1 - 37 . (canceled) 
     
     
         38 . A vector or segment thereof comprising multiple copies of a transcription unit separated by at least one selective marker gene wherein the multiple copies of a transcription unit comprise at least two copies of a transcription unit that encodes an immunoglobulin light chain polypeptide or variable region thereof and at least two copies of a transcription unit that encodes an immunoglobulin heavy chain polypeptide or variable region thereof 
     
     
         39 . The vector or segment thereof of  claim 38  wherein the number of copies of the transcription unit encoding an immunoglobulin light chain polypeptide or variable region thereof is two and the number of copies of the transcription unit encoding an immunoglobulin heavy chain polypeptide or variable region thereof is two. 
     
     
         40 - 42 . (canceled) 
     
     
         43 . The vector or segment thereof of  claim 38  wherein each transcription unit is under the control of its own promoter and 3′ untranslated region. 
     
     
         44 . The vector or segment thereof of  claim 38  wherein each transcription unit is under the control of its own promoter and 3′ untranslated region, and wherein the promoter is an SV40, HSV, bovine growth hormone, thymidine kinase, MPSV, mouse beta globin, human EF1, MSV-LTR, RSV, MMTV-LTR, CMV, MLV, Chinese hamster elongation factor, or mouse Abelson LTR promoter. 
     
     
         45 . The vector or segment thereof of  claim 38  wherein the vector further comprises multiple enhancers. 
     
     
         46 - 50 . (canceled) 
     
     
         51 . The vector or segment thereof of  claim 38  wherein the multiple copies of the transcription unit are in a direct repeat orientation. 
     
     
         52 . The vector or segment thereof one of  claim 38  wherein the multiple copies of the transcription unit are in an inverted repeat orientation. 
     
     
         53 . The vector or segment thereof of  claim 38  wherein the transcription unit is a bi-cistronic or poly-cistronic unit. 
     
     
         54 . The vector or segment thereof of  claim 38  wherein the transcription unit comprises an internal ribosome entry site. 
     
     
         55 - 59 . (canceled) 
     
     
         60 . The vector or segment thereof of  claim 38  wherein the selective marker gene is a gpt, res, neo, his, DHFR, GS, adenosine deaminase (ADA), thymidine kinase (TK), adenine phosphoribosyl transferase (APRT), zeocin resistance, hygromycin resistance or puromycin resistance gene. 
     
     
         61 - 62 . (canceled) 
     
     
         63 . An expression vector or segment thereof according to  claim 38 . 
     
     
         64 . A eukaryotic host cell comprising an expression vector or segment thereof according to  claim 63 . 
     
     
         65 . The host cell according to  claim 64  comprising an additional expression vector or segment thereof according to  claim 63 , said additional expression vector or segment thereof comprising a different selective marker gene. 
     
     
         66 . (canceled) 
     
     
         67 . The host cell according to  claim 64 , wherein the host cell is a plant, insect, mammalian or yeast cell. 
     
     
         68 . The host cell according to  claim 64  wherein the host cell is a Chinese hamster ovary (CHO) cell or CHO-K1 cell. 
     
     
         69 . The host cell according to  claim 64  wherein the expression vector or segment thereof is integrated into the host cell chromosome. 
     
     
         70 . A stable cell line comprising an expression vector or segment thereof according to  claim 63 . 
     
     
         71 . The stable cell line according to  claim 70  wherein the cells are transformed or transfected by at least one and not more than six expression vectors or segments thereof. 
     
     
         72 . A DHFR +  CHO-K1 cell line, DHFR −  DUKX-B11 (DXB11) cell line or DG-44 cell line according to  claim 70 . 
     
     
         73 - 108 . (canceled) 
     
     
         109 . A vector or segment thereof comprising multiple copies of a transcription unit each separated by a selective marker gene wherein the multiple copies of a transcription unit comprise at least two copies of a transcription unit that encodes an immunoglobulin light chain polypeptide or variable region thereof and at least two copies of a transcription unit that encodes an immunoglobulin heavy chain polypeptide or variable region thereof 
     
     
         110 . The vector or segment thereof of  claim 109  wherein the number of copies of the transcription unit encoding an immunoglobulin light chain polypeptide or variable region thereof is two and the number of copies of the transcription unit encoding an immunoglobulin heavy chain polypeptide or variable region thereof is two. 
     
     
         111 - 113 . (canceled) 
     
     
         114 . The vector or segment thereof of  claim 109  wherein each transcription unit is under the control of its own promoter and 3′ untranslated region. 
     
     
         115 . The vector or segment thereof of  claim 109  wherein each transcription unit is under the control of its own promoter and 3′ untranslated region, and wherein the promoter is an SV40, HSV, bovine growth hormone, thymidine kinase, MPSV, mouse beta globin, human EF1, MSV-LTR, RSV, MMTV-LTR, CMV, MLV, Chinese hamster elongation factor, or mouse Abelson LTR promoter. 
     
     
         116 . The vector or segment thereof of  claim 109  wherein the vector further comprises multiple enhancers. 
     
     
         117 - 121 . (canceled) 
     
     
         122 . The vector or segment thereof of  claim 109  wherein the multiple copies of the transcription unit are in a direct repeat orientation. 
     
     
         123 . The vector or segment thereof of  claim 109  wherein the multiple copies of the transcription unit are in an inverted repeat orientation. 
     
     
         124 . The vector or segment thereof of  claim 109  wherein the transcription unit is a bi-cistronic or poly-cistronic unit. 
     
     
         125 . The vector or segment thereof of  claim 109  wherein the transcription unit comprises an internal ribosome entry site. 
     
     
         126 - 130 . (canceled) 
     
     
         131 . The vector or segment thereof of  claim 109  wherein the selective marker gene is a gpt, res, neo, his, DHFR, GS, adenosine deaminase (ADA), thymidine kinase (TK), adenine phosphoribosyl transferase (APRT), zeocin resistance, hygromycin resistance or puromycin resistance gene. 
     
     
         132 - 133 . (canceled) 
     
     
         134 . An expression vector or segment thereof according to  claim 109 . 
     
     
         135 . A eukaryotic host cell comprising an expression vector or segment thereof according to  claim 134 . 
     
     
         136 . The host cell according to  claim 135  comprising an additional expression vector or segment thereof according to  claim 134 , said additional expression vector or segment thereof comprising a different selective marker gene. 
     
     
         137 . (canceled) 
     
     
         138 . The host cell according to  claim 135 , wherein the host cell is a plant, insect, mammalian or yeast cell. 
     
     
         139 . The host cell according to  claim 135  wherein the host cell is a Chinese hamster ovary (CHO) cell or CHO-K1 cell. 
     
     
         140 . The host cell according to  claim 135  wherein the expression vector or segment thereof is integrated into the host cell chromosome. 
     
     
         141 . A stable cell line comprising an expression vector or segment thereof according to  claim 134 . 
     
     
         142 . The stable cell line according to  claim 141  wherein the cells are transformed or transfected by at least one and not more than six expression vectors or segments thereof. 
     
     
         143 . A DHFR +  CHO-K1 cell line, DHFR −  DUKX-B11 (DXB11) cell line or DG-44 cell line according to  claim 141 . 
     
     
         144 . A vector or segment thereof comprising the elements (transcription unit−transcription unit)−selective marker−(transcription unit−transcription unit), wherein two of said transcription units comprise a nucleic acid sequence encoding an immunoglobulin light chain or variable region thereof and two of said transcription units comprise a nucleic acid sequence encoding an immunoglobulin heavy chain or variable region thereof. 
     
     
         145 . The vector or segment thereof of  claim 144  wherein each transcription unit is under the control of its own promoter and 3′ untranslated region. 
     
     
         146 . The vector or segment thereof of  claim 144  further comprising an internal ribosome entry site. 
     
     
         147 . An expression vector or segment thereof according to  claim 144 . 
     
     
         148 . A eukaryotic host cell comprising an expression vector or segment thereof according to  claim 147 . 
     
     
         149 . The host cell according to  claim 148  comprising an additional expression vector or segment thereof according to  claim 147 , said additional expression vector or segment thereof comprising a different selective marker gene. 
     
     
         150 . The host cell according to  claim 148  wherein the host cell is a Chinese hamster ovary (CHO) cell or CHO-K1 cell. 
     
     
         151 . A method of producing a recombinant polypeptide comprising:
 (a) culturing the host cell of  claim 148 ; and   (b) expressing said recombinant polypeptide.