Design of enzyme systems for selective product release from microbial cells; isolation of a recombinant protein from...

The development of expression systems for recombinant proteins and recombinant protein particles that cannot be secreted and that are located in specific cell locations necessitates the development of novel, more selective, techniques for cell disruption. Mechanical cell disruption methods do not discriminate the release of the desired product from among a host of other contaminating molecules and cell debris, and they also may damage the protein product. In contrast, the use of lytic enzyme systems, which can provide biological specificity to the process of cell lysis and disruption, shows an interesting potential for controlled lysis. In this report, the design and the use of lytic enzyme systems for differential product release from microbial cells have been reviewed. Lytic enzyme systems are usually specific either for yeast or for different types of bacteria. Moreover, the activity profile of a lytic system will have an effect on the product distribution. This profile can be manipulated at the genetic, physiological, production reactor, enzyme purification, and lysis reactor levels. Alternative process designs that will allow the sequential release of products from different cell locations have been reviewed and discussed. Alternatives have been explored by process modeling, process simulation, and optimization techniques. These studies show that the use of lytic enzyme systems has tremendous promise as a method of controlled lysis and differential product release. Finally, the release of a specific recombinant protein, human serum albumin (HSA) from yeast cells, has been investigated. The low levels of wall-lytic protease present in the Oerskovia lytic enzyme system have no deleterious effect on the protein product, and the level of HSA extracted from two positive yeast clones using lytic enzymes is similar to that extracted after bead breakage.