Efficient selectivity of recombinant proteins

Abstract

The invention provides a new expression system comprising a mammalian selectable marker that promotes desirable post-translational modifications of glycoproteins. In particular, the invention includes methods and compositions for optimal recombinant protein expression in mammalian cells by employing a selection marker system based on GPT genes of mammalian origin. The invention includes methods that facilitate selectivity and enhanced expression copies as well as protein yield of recombinant proteins in mammalian cells, and methods of using GPT expression systems.

1 .- 29 . (canceled) 
     
     
         30 . A method of employing tunicamycin (Tn) as a selection marker in mammalian cell culture, comprising
 (a) providing a mammalian host cell population,   (b) introducing into the cell population of step (a) a nucleic acid by transfection to permit integration of the nucleic acid into a target locus, wherein the nucleic acid comprises (i) a mammalian tunicamycin (Tn)-resistance gene encoding a protein having at least 93% identity to the amino acid sequence of SEQ ID NO: 3, and (ii) a first a gene of interest (GOI),   (c) culturing the cell population of step (b) in the presence of Tn, and   (d) obtaining a cell transfectant that comprises said nucleic acid integrated in the target locus.   
     
     
         31 . The method of  claim 30 , wherein said integration into the target locus is mediated by a recombinase that recognizes a specific site in the target locus. 
     
     
         32 . The method of  claim 30 , wherein said integration into the target locus is mediated by homologous recombination. 
     
     
         33 . The method of  claim 30 , wherein said homologous recombination is facilitated by a nuclease that cleaves a sequence within the target locus. 
     
     
         34 . The method of  claim 33 , wherein said nuclease is selected from a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or an RNA-guided endonuclease. 
     
     
         35 . The method of  claim 30 , wherein said culturing in step (c) comprises culturing in sequentially increasing concentrations of Tn. 
     
     
         36 . The method of  claim 30 , wherein said culturing in step (c) comprises culturing in the presence of Tn at a first concentration, and wherein the cell transfectant obtained from step (d) is further cultured in the presence of Tn at a second concentration higher than the first concentration. 
     
     
         37 . The method of  claim 30 , wherein the Tn-resistance gene encodes a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. 
     
     
         38 . The method of  claim 37 , wherein the Tn-resistance gene comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. 
     
     
         39 . The method of  claim 30 , wherein the first GOI encodes a first protein of interest (POI). 
     
     
         40 . The method of  claim 39 , further comprising expressing said first POI from said first GOI and isolating said first POI from the cultured cell transfectant. 
     
     
         41 . The method of  claim 30 , wherein the mammalian host cell is selected from the group consisting of CHO, COS-7, HEK293, tumor cell, lymphocyte, retinal cell, and stem cell. 
     
     
         42 . The method of  claim 30 , wherein the mammalian host cell is a CHO cell.