Proteins, due to their complex structures, possess infinite possibilities for assembling themselves as well as other biological and nonbiological materials into complex, multicomponent structures. Many examples from nature demonstrate the high degree of flexibility, strength and “intelligence” offered by protein-organized, self-assembling systems. However, several problems must be overcome before self-assembling protein-based products can become commercially feasible. One problem is the high cost of protein production. A second problem involves the question of producing nonidentical protein subunits in the proper stoichiometry for self-assembly. A third problem involves the stability of the protein components of larger structures.
We have been developing protein-producing bioreactors to address the problems associated with the commercial production of proteins, both as individual products as well as components of self assembling systems. The approach that we have taken is to clone the gene or cDNA that encodes the desired protein(s) and tailor the expression of the protein so that it can be produced at high levels under conditions where cell division is blocked. This assures that supplied nutrients go into product and not biomass, and at the same time provides conditions where cells can be immobilized without fracturing the support matrix during division. The blocking of cell growth also allows for the adjustment of protein stoichiometry by setting the ratios of cells that produce different subunits of complex structures.
To simplify downstream processing and purification, we have designed a system where nearly pure protein is secreted directly into the medium. This approach allows for automated downstream processing and separation of product from cells before it is degraded. The model system that we are using for the development of the protein-producing bioreactors utilizes genetically modified strains of Escherichia coli that secrete proteins which are usually found in the periplasmic space directly into the medium. The phosphatebinding protein serves as an ideal model protein for bioreactor development, since the regulatory elements that control its production are turned on by phosphate limitation, which also arrests cell division. We are presently determining if these regulatory and secretory elements can be used to direct the synthesis and secretion of heterologous proteins.