Abstract
Faced with the current wealth of genomic data, it is essential to have robust and reliable methods of converting DNA sequences into their functional gene products. We demonstrate here that when conditions are established that take advantage of the replication-associated virus amplification, the virus-induced shutdown of host protein synthesis as well as the activation of signalling pathways that normally occur during virus replication, adenovirus biology can be exploited to generate a potent kinase expression system. Residual virus in the protein production has always been a limitation for adenovirus systems and we describe a DNA intercalator/ultraviolet light treatment that eliminates residual adenovirus in protein preparations that has no deleterious effect on enzyme activity. The use of mammalian cells in combination with adenovirus generated a variety of active enzymes which could not be produced in Escherichia coli or baculovirus-infected insect cells. Thus, the utility of adenovirus-mediated enzyme expression as a versatile alternative to established protein production technologies is demonstrated.
Highlights
Amidst the current abundance of new genomic data, it becomes increasingly important to have reliable methods of converting these DNA sequences into their functional gene products
We describe here optimized expression conditions combined with a simple onestep af®nity puri®cation to generate 100 mg to 10 mg quantities of enzymatically active mammalian protein kinases that could not be produced by either bacterial or baculovirus systems
Most uses of recombinant adenovirus apply the virus to cells that do not support virus replication
Summary
Amidst the current abundance of new genomic data, it becomes increasingly important to have reliable methods of converting these DNA sequences into their functional gene products. A small subset of the mammalian kinome, for example, [1] has been studied at the biochemical level and a full understanding of the biology of these enzymes will require methods to generate functional proteins. As increasing numbers of the more exotic mammalian kinases are examined, it becomes clear that expression of these proteins in either prokaryotic or insect cells does not always result in soluble and functional enzymes. Some of these failures can be attributed to the requirement for mammalian speci®c chaperones, co-factors or post-translational modi®cation (e.g. phosphorylation, glycosylation) that are missing in the bacterial or insect cells. Methods to overexpress and purify mammalian enzymes using mammalian cells can be of substantial value
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