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        Expression of Recombinant Membrane-Type MMPs

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        Since the discovery of the membrane-type matrix metalloproteinases (MT-MMPs) which are characterized by the existence of a C-terminal membrane-spanning domain followed by a short cytoplasmic “tail” (1 ) there has been much interest in the production of recombinant protein to facilitate both structural and functional analyses. The proteolytic activity of the membrane-type-1 MMP (MT1-MMP) has been studied using recombinant material produced from expression in E. coli (2 ,3 ). These data have been confirmed and extended by reports describing expression of MT1-MMP in mammalian cells (4 7 ). This chapter will, therefore, focus on methods to produce recombinant MT1-MMP in both types of expression systems and the issues governing the choice of which system to use will be discussed. Overall, we have used the glutathione-S-transferase (GST)-fusion technology to produce material for injection into sheep to raise polyclonal antibodies. For kinetic analysis of catalytic activity we have used E. coli expression, both periplasmic, and expression in inclusion bodies (followed by solu-bilization and refolding). We have found the latter approach to be most reliable and, therefore, describe this method in detail. To study the location and function of the membrane-associated form we have used transient expression in Chinese hamster ovary (CHO) cells. High level expression in stably trans-fected cells has been more difficult to achieve and this chapter describes the use of an inducible mammalian cell system which gives reasonably high levels of expression of MT1-MMP.
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