Protein production systems, in which polypeptides or proteins of interest are produced in recombinant organisms or cells, are the backbone of commercial biotechnology. The earliest systems, based on bacterial expression in hosts such as E. coli, have been joined by systems based on eukaryotic hosts, in particular mammalian cells in culture, insect cells both in culture and in the form of whole insects, and transgenic mammals such as sheep and goats.
Prokaryotic cell culture systems are easy to maintain and cheap to operate. However, prokaryotic cells are not capable of post-translational modification of eukaryotic proteins. Moreover, many proteins are incorrectly folded, requiring specific procedures to refold them, which adds to the cost of production.
Eukaryotic cell culture systems have been described for a number of applications. For example, mammalian cells are capable of post-translational modification, and generally produce proteins which are correctly folded and soluble. The chief disadvantages of mammalian cell systems include the requirement for specialised and expensive culture facilities, the risk of infection, which can lead to loss of the whole culture, and the risk of contaminating the end product with potentially hazardous mammalian proteins.
Insect cells are also used for polypeptide expression. The most widespread expression system used in insect cells is based on baculovirus vectors. A baculovirus expression vector is constructed by replacing the polyhedrin gene of baculovirus, which encodes a major structural protein of the baculovirus, with a heterologous gene, under the control of the strong native polyhedrin promoter. Cultured insect host cells are infected with the recombinant virus, and the protein produced thereby can be recovered from the cells themselves or from the culture medium if suitable secretion signals are employed. These systems also, however, suffer from problems associated with reproducibility of expression level and quality, infection of the culture, and may require specialised culture facilities. Furthermore, baculovirus stocks, which for the production of certain proteins may have to be made under GMP conditions, is not always stable over time.
Suitable promoters used in Drosophila melanogaster S2 cells for protein expression also include the pMT promoter and the P2ZOp2F (OPIE2 promoter)
Chung et al. Molecular and Cellular Biology, Vol. 10, No. 12, 1992 relates to characterization of positive and negative regulatory elements in the Actin5C distal promoter.
Angelichio et al. Nucleic Acids Research, Vol. 19, No. 18 5037-5043, 1991 relates to a comparison of several promoters and polyadenylation signals for use in heterologous gene expression in cultured Drosophila cells.
It is an object of the present invention to provide more efficient expression and/or secretion in host cells, notably in Drosophila melanogaster. It is a further object to provide polynucleotides and vectors that facilitate this efficient expression and secretion.