The use of bi-cistronic transfer vectors for the baculovirus expression system

Abstract

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.

Introduction

The baculovirus expression vector system (BVES) is widely applicable as an alternative to prokaryotic or other eukaryotic expression systems for the production of high level recombinant proteins. Many recombinant proteins were expressed using this system and in most cases high levels of biologically active products were achieved (for review see Miller, 1988, Summers and Smith, 1988, Luckow, 1990, O’Reilly et al., 1992). Recombinant proteins are usually produced by replacing the viral polyhedrin gene with a foreign gene encoding for the protein of interest. The polyhedrin structural gene is a very late gene that is highly expressed in infected insects but is not essential for viral propagation in cultured insect cells (Smith et al., 1983). In principal, a gene of interest is cloned into a transfer vector under the strong Polyhedrin Promoter (Ppol). This vector also contains flanking sequences of the polyhedrin gene which enable specific homologous recombination to the wild-type baculovirus when the naked DNAs are cotransfected into insect cells. Isolation of recombinant viruses is tedious since homologous recombination is a rare event in transfected insect cells (about 5%). Several approaches were developed in an attempt to simplify and shorten the procedure for efficient isolation of recombinant viruses. One approach utilizes linearized viruses, in which an essential gene for viral propagation is deleted. A viable recombinant virus is produced in transfected cells only upon homologous recombination between a transfer vector that contains the deleted gene and the gene of interest (Kitts, 1995). Another approach to detect a recombinant virus utilizes a reporter gene, β-galactosidase, which is incorporated into the transfer vector leading to the formation of blue plaques only by recombinant viruses (Vialard et al., 1990). Yet, all these improvements of the BVES do not enable an immediate evaluation of the recombinant protein level produced in infected cells and do not allow the on-line monitoring of recombinant protein production in large-scale reactors in real time.

Our goal was to construct bi-cistronic vectors for the BVES that will enable instant evaluation of recombinant protein level at any given time. In general, a gene of interest and a reporter gene are cloned under the same promoter and as such are the products of the same transcriptional unit. The first cistron is translated by the ribosomal scanning mechanism (Kozak, 1987) while the second cistron is translated either via re-initiation of ribosomes or through internal ribosome binding site (reviewed in Mathews, 1996). As a result, a constant ratio between the expression of the first cistron and the second cistron is maintained. An element termed internal ribosome entry site (IRES), borrowed from a picornavirus, serves as an internal binding site for ribosome through a mechanism that does not involve scanning from the 5′ of the bi-cistronic mRNA (Jang et al., 1989). The incorporation of IRES elements in various mammalian expression vectors enabled the co-expression of at least two genes from the same transcript (Morgan et al., 1992, Dirks et al., 1993).

In this communication, the construction of bi-cistronic transfer vectors for the BVES is described. Two reporter genes, chloramphenicol acetyl transferase (CAT) and firefly luciferase (LUC), were cloned downstream to the Ppol. These constructs enabled a strong expression of the gene closer to the Ppol and with about 25-fold reduction in the activity if the same gene is placed as a second cistron. Our results suggest that an efficient re-initiation of ribosomes occurs in insect cells. In some of the constructs the encephalomyocarditis virus (EMCV) IRES element was cloned in between the two reporter genes. Unexpectedly, the presence of the well known efficient IRES element did not enhance the translation of the second cistron but rather led to an additional 50-fold decrease in its translation compared to its activity as first cistron. The fact that the reporter gene, placed as the second cistron in the transfer vectors, was easily detected either in the presence or absence of an IRES element enabled a simple isolation of recombinant viruses expressing the highest level of the gene of interest. Since both cistrons are expressed from the same transcript, a constant ratio between the levels of the two expressed proteins was observed during a recombinant bi-cistronic viral propagation. This facilitated on-line monitoring of the target recombinant protein levels in real time. Therefore, these new bi-cistronic transfer vectors provide new features to the existing BVES vectors.