Production of Active Mammalian and Viral Proteases in Bacterial Expression Systems

Mammalian endopeptidases and exopeptidases participate in a wide variety of cellular processes. They are responsible for the relatively non-specific degradation of proteins targeted for digestion or recycling, and they also perform highly specific single-site cleavages necessary for the activation or inactivation of functional proteins and peptides. Likewise, numerous viruses that infect mammalian cells utilize virusencoded proteases to regulate their replication cycle. Malnmalian proteases are expressed as enzymatically inactive zymogens requiring specific coor post-translational processing by self or other proteases. Virus...encoded proteases are expressed as part of viral polyproteins that also require specific autoprocessing to release the fully active protease. Thus, the sanle theme is used, where structural motifs prevent the enzyme from being active before the appropriate time and place, and catalytic proficiency is regulated by the formation of the active protease (Babe and Craik, 1997). This theme must be kept in mind when designing heterologous expression systems for mammalian and viral proteases to ensure the production of active or activatable enzymes. Advances in the study of proteases in the past two decades have been largely dependent on the ability of researchers to produce significant quantities of pure enzymes. Generally, recombinant gene expression systems have been used to accomplish this task, especially for proteases that are naturally produced in very limited amounts. Heterologous expression systems also have the advantage of being able to produce variant proteases at will, allowing the study of structure-function relationships and modifications of their properties. In addition to basic research, the production of recolnbinant proteases has been crucial to the development of cOlnmercial products. For example, recombinant bovine chyl11osin. an aspaitic protease, is used in the manufacture of cheese, while