The use of bi-cistronic transfer vectors for the baculovirus expression system.
In this communication, we describe the construction of bi-cistronic transfer vectors for the baculovirus expression system (BVES), which are advantageous over the existing vectors. The new vectors provide a simple way to isolate recombinant viruses. More specifically, the gene of interest and the reporter gene luciferase (LUC), constitute the first and second cistrons, respectively, of the same transcript. Therefore, the LUC activity measured during infection of such a bi-cistronic virus, permits an on-line estimation of the recombinant protein level, a very useful feature for large-scale production of recombinant proteins. To achieve expression of the second cistron, the internal ribosome entry site (IRES) element of the encephalomyocarditis virus (EMCV) was employed. However, this element, which is highly efficient in mammalian systems, did not promote efficient internal translation of the second cistron in various insect cells lines originating from different insect species. The lack of efficient internal translation was not due to baculovirus propagation since the same phenomenon was also observed in a viral-free expression system. It seems that a component essential for efficient EMCV IRES activity is either missing or present in limiting amount in insect cells or not compatible. Nevertheless, LUC placed downstream to the IRES element, or immediately downstream to the first cistron, was expressed to a level that enabled the biotechnological application it was designed for.
Finkelstein
Y
Y
Department of Food Engineering & Biotechnology, Technion, Haifa, Israel.
Faktor
O
O
Elroy-Stein
O
O
Levi
B Z
BZ
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D005796:Genes / Q000235:genetics*; D017930:Genes, Reporter; D005822:Genetic Vectors; D007313:Insecta; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D018411:Spodoptera; D014162:Transfection
D017931:DNA Primers; D011994:Recombinant Proteins; D008156:Luciferases