Abstract
The vaccinia virus/bacteriophage T7 expression system was adapted to Chinese hamster ovary (CHO) cells. Vaccinia virus undergoes abortive infection in CHO cells, which is characterized by a sharp reduction in protein synthesis at the stage of viral intermediate gene expression. We determined that expression of a T7 promoter-regulated chloramphenicol acetyltransferase gene was at least 20 times more efficient in permissive BS-C-1 than in CHO cells. The encephalomyocarditis virus 5'-untranslated region, which confers cap-independent translatability to mRNA, stimulated recombinant protein synthesis by 10-fold in both cell lines, maintaining the advantage of the BS-C-1 cells over CHO cells. Since the cowpox virus hr gene overcomes vaccinia virus host range restriction in CHO cells, we constructed a recombinant virus that carries an intact hr gene in addition to the T7 RNA polymerase gene. With this virus, synthesis of T7 RNA polymerase was enhanced and production of a recombinant protein occurred in CHO cells at the level observed in permissive cell lines. Extension of the vaccinia virus/bacteriophage T7 expression system to CHO cells should be of wide interest, as these cells have advantages for preparation of recombinant proteins in research and biotechnology.
Highlights
The vaccinia virus/bacteriophage T7 expression system was adapted to Chinese hamster ovary (CHO) cells
Recombinant Protein Synthesis in CHO Cells—We measured the impact of host range restriction of vaccinia virus (VV) in CHO cells on expression of a target gene using the VV/T7 system
Permissive (BS-C-1) or nonpermissive (CHO) cells were coinfected with 15 pfu/cell of vTF7–3 [3], which encodes the T7 RNA polymerase gene regulated by the vaccinia virus early/late promoter (P7.5) and with vT7CAT, which has a T7 promoter-regulated CAT gene
Summary
The vaccinia virus/bacteriophage T7 expression system was adapted to Chinese hamster ovary (CHO) cells. Since the cowpox virus hr gene overcomes vaccinia virus host range restriction in CHO cells, we constructed a recombinant virus that carries an intact hr gene in addition to the T7 RNA polymerase gene With this virus, synthesis of T7 RNA polymerase was enhanced and production of a recombinant protein occurred in CHO cells at the level observed in permissive cell lines. A successful mammalian expression system that exploits the bacteriophage T7 RNA polymerase to transcribe T7 promoterregulated target genes was described using vaccinia virus (VV) as the vector [1] In this system, recombinant VV carries an integrated T7 polymerase gene regulated by a viral promoter. We constructed a new recombinant VV containing both the CHO hr and the bacteriophage T7 RNA polymerase genes, which allows comparable levels of expression in CHO and fully permissive cell lines. Expression was further enhanced by incorporating the EMCV untranslated leader sequence
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