Efficient expression from one CMV enhancer controlling two core promoters.
The human CMV promoter/enhancer is one of the strongest promoters for recombinant protein expression in mammalian cells, making the promoter very popular for production of recombinant antibodies. We used an antibody vector design where the antibody heavy and light chain genes were transcribed from a promoter complex consisting of two promoters arranged divergently with the 5' ends of the promoters in close proximity. However, when two identical CMV promoters constituted this promoter complex, the antibody expression observed was lower than expected based on the strength of the individual promoters. To optimize expression we prepared truncated promoter complexes where only one CMV enhancer controlled the initiation of transcription from two divergent minimal CMV core promoters. Antibody expression from the truncated promoter complexes was analyzed both when transiently transfected and upon stable site-specific integration into a CHO DG44 derived cell line. The data showed that it was possible for one enhancer to drive the expression of two core promoters. However, efficient expression from both divergent core promoters was seen only when the unique region upstream of the CMV enhancer was removed. Notably, a 12-fold increase in expression was found from the best of the truncated promoter complexes after stable site-specific integration when compared to the full-length double CMV promoter complex.
Symphogen A/S, Elektrovej Building 375, 2800, Lyngby, Denmark. cra@symphogen.com
Christina Rottbøll
CR
Andersen
Lars Søegaard
LS
Nielsen
Alexandra
A
Baer
Anne Bondgaard
AB
Tolstrup
Dietmar
D
Weilguny
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003587:Cytomegalovirus / Q000235:genetics*; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000469:Alkaline Phosphatase