Efficient Expression from One CMV Enhancer Controlling Two Core Promoters

• Published in: Molecular biotechnology Vol. 48; no. 2; pp. 128 - 137
• Main Authors: Andersen, Christina Rottbøll, Nielsen, Lars Søegaard, Baer, Alexandra, Tolstrup, Anne Bondgaard, Weilguny, Dietmar
• Format: Journal Article
• Language: English
• Published: New York Springer-Verlag 01.06.2011; Humana Press Inc; Springer; Springer Nature B.V
• Subjects: Alkaline Phosphatase - metabolism; Animals; Antibodies; Biochemistry; Biological and medical sciences; Biological Techniques; Biotechnology; Cell Biology; Cellular biology; Chemistry; Chemistry and Materials Science; CHO Cells; Cricetinae; Cricetulus; Cytomegalovirus; Cytomegalovirus - genetics; Data processing; Enhancer Elements, Genetic - genetics; Enhancers; Enzyme-Linked Immunosorbent Assay; Fundamental and applied biological sciences. Psychology; Gene expression; genes; Genetic Vectors - genetics; Human cytomegalovirus; Human Genetics; Humans; Immunoglobulins; Integration; Light chains; Mammalian cells; Promoter Regions, Genetic - genetics; Promoters; Protein Science; protein synthesis; Proteins; recombinant antibodies; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleic acid; RNA; Transcription; Transcription initiation; CMV enhancer; Minimal CMV promoter; Chinese hamster ovary (CHO); mRNA; Antibody expression; Promoter complex; Messenger RNA; Antibody; Complexes; CHO cell line
• ISSN: 1073-6085; 1559-0305
• DOI: 10.1007/s12033-010-9353-7

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.