title: High levels of protein expression using different mammalian CMV promoters in several cell lines.
abstract: With the recent completion of the human genome sequencing project, scientists are faced with the daunting challenge of deciphering the function of these newly found genes quickly and efficiently. Equally as important is to produce milligram quantities of the therapeutically relevant gene products as quickly as possible. Mammalian expression systems provide many advantages to aid in this task. Mammalian cell lines have the capacity for proper post-translational modifications including proper protein folding and glycosylation. In response to the needs described above, we investigated the protein expression levels driven by the human CMV in the presence or absence of intron A, the mouse and rat CMV promoters with intron A, and the MPSV promoter in plasmid expression vectors. We evaluated the different promoters using an in-house plasmid vector backbone. The protein expression levels of four genes of interest driven by these promoters were evaluated in HEK293EBNA and CHO-K1 cells. Stable and transient transfected cells were utilized. In general, the full-length human CMV, in the presence of intron A, gave the highest levels of protein expression in transient transfections in both cell lines. However, the MPSV promoter resulted in the highest levels of stable protein expression in CHO-K1 cells. Using the CMV driven constitutive promoters in the presence of intron A, we have been able to generate >10 microg/ml of recombinant protein using transient transfections.
authors.affiliation: Systems Biology, Berlex Biosciences, Richmond, CA, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Xia
authors.forename: Peter
authors.initials: P
authors.lastname: Bringmann
authors.forename: John
authors.initials: J
authors.lastname: McClary
authors.forename: Patrick P
authors.initials: PP
authors.lastname: Jones
authors.forename: Warren
authors.initials: W
authors.lastname: Manzana
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhu
authors.forename: Soujuan
authors.initials: S
authors.lastname: Wang
authors.forename: Yi
authors.initials: Y
authors.lastname: Liu
authors.forename: Susan
authors.initials: S
authors.lastname: Harvey
authors.forename: Mary Rose
authors.initials: MR
authors.lastname: Madlansacay
authors.forename: Kirk
authors.initials: K
authors.lastname: McLean
authors.forename: Mary P
authors.initials: MP
authors.lastname: Rosser
authors.forename: Jean
authors.initials: J
authors.lastname: MacRobbie
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Olsen
authors.forename: Ronald R
authors.initials: RR
authors.lastname: Cobb
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003587:Cytomegalovirus / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D051379:Mice; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D009053:Sarcoma Viruses, Murine / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection
chemical_list: D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins