Stably Maintained Multiple Copies of at Least Two Orf in the Same Orientation

Abstract

The present invention relates to a method for constructing a host cell expressing a polypeptide of interest from at least two ORF's stably integrated onto the chromosome of the host cell comprising the steps of: a) providing the at least two ORF's encoding the same polypeptide, wherein the at least two DNA sequences of the ORF's differ in at least one position; b) integrating the at least two ORF's in the same orientation on the host cell chromosome.

1 - 22 . (canceled)  
     
     
         23 . A method for constructing a host cell expressing a polypeptide of interest from at least two open reading frames stably integrated onto the chromosome of the host cell comprising the steps of: 
 a) providing the at least two open reading frames encoding the same polypeptide, wherein the at least two DNA sequences of the open reading frames differ in at least one position;    b) integrating the at least two open reading frames in the same orientation on the host cell chromosome.    
     
     
         24 . The method of  claim 23 , wherein the at least two open reading frames are integrated in the same location on the host cell chromosome.  
     
     
         25 . The method of  claim 23 , wherein the open reading frames are transcribed as a polycistronic mRNA.  
     
     
         26 . The method of  claim 23 , wherein segments of 100% sequence identity between the open reading frames are at the most 1000 bp.  
     
     
         27 . The method of  claim 23 , wherein segments of 100% sequence identity between the open reading frames are at the most 80 bp.  
     
     
         28 . The method of  claim 23 , wherein the open reading frames encode an enzyme.  
     
     
         29 . The method of  claim 28 , wherein the enzyme comprises a protease.  
     
     
         30 . The method of  claim 29 , wherein the protease is a serine protease.  
     
     
         31 . The method of  claim 30 , wherein the serine protease is chosen from the group consisting of protease family S2A or S1E.  
     
     
         32 . The method of  claim 31 , wherein the serine protease is encoded from a sequence chosen from the group consisting of Seq ID NO. 1, 2, or 51.  
     
     
         33 . A host cell comprising at least two open reading frames encoding the same polypeptide stably integrated in the same orientation onto the host cell chromosome wherein the at least two DNA sequences of the open reading frames differ in at least one position.  
     
     
         34 . The host cell of  claim 33 , wherein the at least two open reading frames are integrated in the same location on the host cell chromosome.  
     
     
         35 . The host cell of  claim 34 , wherein the open reading frames are transcribed as a polycistronic mRNA.  
     
     
         36 . The host cell of  claim 33 , wherein segments of 100% sequence identity between the open reading frames are at the most 1000 bp.  
     
     
         37 . The host cell of  claim 33 , wherein segments of 100% sequence identity between the open reading frames are at the most 80 bp.  
     
     
         38 . The host cell of  claim 33 , wherein the open reading frames encode an enzyme.  
     
     
         39 . The host cell of  claim 38 , wherein the enzyme comprises a protease.  
     
     
         40 . The host cell of  claim 39 , wherein the protease is a serine protease.  
     
     
         41 . The host cell of  claim 40 , wherein the serine protease is chosen from the group consisting of protease family S2A or S1E.  
     
     
         42 . The host cell of  claim 41 , wherein the serine protease is encoded from a sequence chosen from the group consisting of Seq ID NO. 1, 2, or 51.  
     
     
         43 . A nucleic acid construct comprising at least two open reading frames encoding the same polypeptide in the same orientation, wherein the at least two DNA sequences of the open reading frames differ in at least one position.  
     
     
         44 . A method of producing the same polypeptide from at least two open reading frames stably integrated onto the host cell chromosome comprising the steps of: 
 i) culturing the host cell of  claim 33  under conditions conducive for the expression of the polypeptide; and optionally    ii) recovering the polypeptide.