Insect expression systems : improving intracellular and heterologous gene expression
Expression systems (ES) in a wide variety of biological systems are used to provide foreign protein. Protein production in mammalian cells is a labour-intensive and expensive process and insect cells have been used as cheaper alternatives. Insect ES are based on two types of vector; viral- and plasmid-based. The baculovirus ES (BES) offers high-level transient production of intracellular proteins. Yields of secreted and membrane-targeted proteins have until recently been relatively low, however, the development of a novel vector, lacking chitinase, has overcome these limitations. Plasmid-based vectors can be used for transient and stable expression in insect cells but most utilise the relatively weak Autographa californica multiple nucleopolyhedrovirus (AcMNPV) immediate early-1 (ie-1) promoter, producing low-levels of intracellular recombinant protein. More recent vectors have used stronger promoters, however, compared with mammalian stable ES these are limited in use and most notable is the lack of an efficient inducible ES in insect cells. The work in this thesis describes methods, with which intracellular levels of foreign protein may be increased, while also developing the grounding work for the development of a fully functional insect tetracycline regulatory system. To improve stable and regulated expression of heterologous genes in insect cells, work was undertaken to compare and characterise the transcriptional activity of a range of promoters successfully used in insect and mammalian systems. Of the promoters tested, Orgyia pseudotsugata MNPV (Op) ie-2 and Bombyx morl actin 3 (BmA3+E) were found to be transcriptionally stronger than AcMNPV ie-1. Drosophila melanogaster metallothionein and actin 5.1 did not produce any detectable activity, and the Cytomegalovirus (CMV) ie promoter, although active, was weaker than AcMNPV ;e-1. Attempts to develop a new inducible ES for use in stable cell lines was based on modifying a mammalian tetracyclineinducible ES. The original CMVie promoters were replaced by the Bm A3+E and Op ie-2 promoters that had been shown to be optimal in insect cells. The Bm A3+E promoter was successfully used to express the tetracycline transcriptional activator protein. Studies also demonstrated that the constitutive transcriptional activity of Op ie-2, used to drive expression of a reporter gene, was successfully suppressed in normal medium. However, in the presence of the inducer, doxycycline, transcriptional activity of Op ie-2 was not activated. Work to elucidate why Op ie-2 remain repressed in the presence of the inducer, indicated that other, uncharacterised vector sequences may have interfered with the activation process. To determine whether stable insect cell lines could be used as an effective alternative to the BES for producing large quantities of intracellular foreign proteins, protein production from stable cell lines, using the Bm A3+E promoter, were compared to the BES using a range of reporter proteins. It was concluded that with vectors currently available, stable cell lines would not normally provide an effective alternative to the BES. However, a stable insect cell line expressing Discosoma red was used to develop methods for scaling-up continuous cultivation of cells in an open fermenter system. This stable cell line was successfully maintained without contamination or total loss of cell viability for 4 weeks. Improving intracellular expression of foreign genes using the BES, initially focused on investigating whether a baculovirus vector (BV) lacking chitinase could produce larger yields than normal BV. It was concluded that recombinant BV lackingchitinase could be used to improve intracellular levels of foreign protein, but demonstrated the importance of optimiSing production conditions for each recombinant protein (e.g., vector and cell line). Intracellular levels of foreign protein were further improved from the BES using different culturing methodologies (fermentation and shaker flask) and concluded that fermenters produced optimal conditions for intracellular protein production, probably due to maintaining a constant level of dissolved oxygen concentration during virus infection.
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