Internal ribosome entry sites for recombinant protein expression

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The invention describes compositions and methods for recombinant protein expression in a wide range of cell types, including mammalian, insect, and bacterial cells. The compositions comprise a viral IRES sequence selected from enterovirus 71 (EV71), hepatitis C virus (HCV), or encephalomyocarditis virus (EMCV), or a variant or fragment thereof, or alternatively, a homolog of a viral IRES selected from EV71, HCV, or EMCV, or a variant or fragment thereof. Methods of using the compositions are also described.

The present invention relates to the 5′untranslated regions (5′UTRs) of viral genes which function as internal ribosome entry sites (IRESs). In particular, the present invention relates to the IRES of encephalomyocarditis virus (EMCV), Hepatitis C virus (HCV), and Enterovirus 71 (EV71). The present invention further relates to methods of using the various IRESs in recombinant protein expression systems, to compositions comprising the various IRESs, and to methods of screening for anti-viral compounds using the IRESs of the present invention.

Eukaryotic mRNAs have a distinctive structural feature at their 5′end, called a5′cap, which is a residue of 7-methylguanosine linked to the 5′terminal residue of the mRNA through an unusual 5′,5′-triphosphate linkage. Cap-dependent translation is initiated by the binding of the cap-binding protein complex eIF-4F to the 5′cap, which in turn facilitates the binding of the 43S ternary ribosomal subunit near or at the 5′cap region. The ribosome complex is purported to scan the mRNA from the 5′ cap until it encounters the first AUG initiation codon, where translation of the mRNA is initiated.

Contrary to the above reports, the inventors have surprisingly found that the EMCV IRES element functions in baculovirus host insect cells. The inventors have also found other IRESs that function in baculovirus host insect cells as well as in other cell types, including mammalian and bacterial cells. Thus, the present invention provides a kit for recombinant protein expression in bacteria, insect, and/or mammalian cells comprising at least one nucleic acid vector comprising at least one IRES sequence functional in a bacterial cell, at least one nucleic acid vector comprising at least one IRES sequence functional in a insect cell, and at least one nucleic acid vector comprising at least one IRES sequence function in a mammalian cell.

The present invention also provides homologs, fragments, and variants of the IRESs of EV71, HCV, and EMCV, as well as variants and fragments of homologs of the EV71, HCV, and EMCV IRESs. The present invention further provides multicistronic nucleic acid vectors comprising a viral IRES disclosed in the present invention or a homolog, fragment, or variant thereof having IRES activity, for the production of multiple recombinant proteins from a single mRNA transcript. These multicistronic nucleic acid vectors may be contained in a biological vector capable of expressing multiple genes in a host cell. These nucleic acid vectors and biological vectors may be used for the genetic treatment in patients and/or the recombinant proteins produced thereby may be useful as therapeutic agents.

The present invention also provides a baculovirus transfer vector and a recombinant baculovirus for the expression of at least two genes in a baculovirus host cell, comprising a viral IRES disclosed in the present invention or a homolog, variant, or a fragment thereof having IRES activity. The ability to express two or more genes from a single baculovirus transfer vector and a recombinant baculovirus greatly simplifies the process of isolating plaques expressing the gene(s) of interest. Moreover, the expression of a gene of interest and a reporter gene would also allow the simultaneous evaluation of recombinant protein level produced and the detection/isolation of cells producing the recombinant protein.