Expression of MMPs andTIMPs in Mammalian Cells

Abstract

Expression of recombinant matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in mammalian cells is an important step in their functional characterization. Also, transient transfection analysis of promoter constructs driving CAT or luciferase reporter genes is a mainstay of gene regulation studies. One of the critical advantages of mammalian expression over bacterial systems for production of functional proteins is that problems of refolding, which are particularly significant for the MMPs and TIMPs, are generally not encountered. Transient expression in COS cells is very rapid and can be useful in many situations, for instance to demonstrate activity or to compare the characteristics of mutant proteins. It can also generate sufficient quantities of protein for biochemical and cell biological studies, but most often bulk production will require stable expression. In this chapter we will discuss our experiences with transient expression in COS-1 and C3H 10T1/2 mouse fibroblasts, and describe two systems that we have used for constitutive stable expression, namely BHK (Baby Hamster Kidney) and the NS0 myeloma cell line. This chapter complements the information in Chapter 11 by Butler, d’Ortho, and Atkinson who have described stable, tetracycline-regulated expression of MT1-MMP in CHOL cells. Also, the reader should note that many expression vector systems are now available commercially, and we have not attempted here to provide comparisons.