9] Cell-free synthesis of noninterstitial (CHL cell) procollagen chains

Abstract

This chapter presents the methodologies employed to achieve the cell-free synthesis of procollagen chains directed by components of Chinese hamster lung (CHL) cells, which synthesize a noninterstitial collagen. Each of the three cell-free systems described in this chapter offers unique opportunities for investigating the biosynthesis and metabolism of noninterstitial procollagen chains. The CHL S30-catalyzed system is an homologous cell-free system, and as a consequence, any positive or negative translational element(s), i.e., specific tRNA or initiation factor, involved in noninterstitial procollagen chain mRNA expression is presented. The CHL polysome-directed system offers the advantage of allowing the discrimination among transcribed but not translated procollagen chain mRNA species from those that are actively being expressed. The heterologous cell-free protein synthesis system programmed by CHL mRNA offers the opportunity not only to discern positive translational regulatory elements but also to identify factors and enzymes involved in the posttranslational modification(s) and catabolism of a noninterstitial procollagen chain.