Requirements for optimal expression of secreted and nonsecreted recombinant proteins in vaccinia virus systems.
Selection of an optimal promoter is necessary for efficient expression of foreign genes with vaccinia virus. Since a variety of powerful (homologous) vaccinia virus promoters and foreign (heterologous) promoter systems have been described for use in vaccinia, we have addressed the question of whether a general rule exists that allows the prediction of the optimal promoter/gene combination. We have compared the expression properties of four secreted proteins, the human blood clotting factor IX (FIX), the human blood glycoprotein Protein S (ProtS), the human von Willebrand factor (vWF), and the Hepatitis B virus (HBV) middle surface glycoprotein preS2, with proteins that were reported not to be secreted, the HBV large surface glycoprotein preS1 and the murine leukemia virus (MuLV) BM-5 Eco gag protein. In addition, we have included in our study an internal control protein, the vaccinia virus p11 protein, to monitor possible side effects of the promoter system used. Genes encoding the foreign proteins were placed either under control of a synthetic vaccinia virus early/late promoter (selP) or under control of the bacteriophage T7 promoter (T7/emc system). The secreted proteins were more efficiently expressed when fused to the homologous promoter. Direct comparison of the two promoters indicated that the expression level ranged between 1.4 (ProtS) and 3.9 (FIX)-fold higher with the selP than with the T7 promoter. In contrast, the cell-associated HBV preS1 was more efficiently expressed under the T7 promoter and the MuLV BM-5 Eco gag polypeptide was expressed equally well from both promoters. These data indicate that a careful prediction of optimal promoter/foreign gene combinations for the vaccinia virus expression system is possible. The choice of the optimal promoter/expression system is based on a simple classification scheme, discriminating secreted and nonsecreted proteins.
Immuno AG Biomedical Research Center, Orth/Donau, Austria.
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Pfleiderer
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Falkner
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Dorner
D000818:Animals; D017123:Bacteriophage T7; D001483:Base Sequence; D002460:Cell Line; D003593:Cytoplasm; D005164:Factor IX / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression; D015683:Gene Products, gag / Q000096:biosynthesis / Q000235:genetics; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D000882:Haplorhini; D006515:Hepatitis B virus / Q000737:chemistry; D006801:Humans; D009052:Leukemia Virus, Murine / Q000737:chemistry; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D017293:Protein S / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000145:classification / Q000235:genetics; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics; D014841:von Willebrand Factor / Q000096:biosynthesis / Q000235:genetics
D015683:Gene Products, gag; D006023:Glycoproteins; D017293:Protein S; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D014841:von Willebrand Factor; D005164:Factor IX