The secretion of soluble proteins and the movement of membrane proteins to the cell surface is an important process with which cells regulate a plethora of different functions, such as intercellular communication, hormonal regulation, signal transfer between synapses, homeostasis, host defence against pathogens etc. Controlling protein secretion with synthetic biological systems thus represents an attractive tool to regulate different biological processes and for use in therapeutic applications. Currently, most synthetic systems for regulated secretion from mammalian cells work by inducing the expression of the secreted protein, which then travel through the conventional secretory patway to the cells exterior. However, in this approach, the protein must first be transcribed and translated, before it can enter the secretory pathway and start to accumulate in the cells exterior, a process which can take several hours before the protein is secreted in biologically relevant amounts. For certain applications, especially therapeutic, where a response needs to occur within minutes, not hours, this delay in response makes secretions with such systems too slow for practical use.
In the present doctoral dissertation we have developed two systems – lumER and membER – for regulated secretion of soluble and membrane proteins, in which secretion is controlled by inducible proteases. In both systems the secreted protein is expressed and retained in the conventional secretory pathway in the endoplasmic reticulum, with the help of appended C-terminal retention sequences. The removal of the retention sequence is under the control of orthogonal viral proteases from the Potyviridae family, whose activity is regulated by small molecules. Because secretion is regulated at the posttranslational level, both systems bypass the need for prior protein synthesis and are therefore faster than comparable systems based on regulating protein expression. We have shown that both systems are orthogonal to each other and are controllable with different orthogonal and inducible viral proteases. Both systems allow for repeated induction of secretion. Further, we have shown that secretion with the membER and lumER systems can be controlled by constructs used for two input logical operation processing. Finally, we have used the systems to control the secretion of therapeutic proteins.
Because ER retention and secretion are achieved with the help of modular elements, the systems allow for their combination with other synthetic and natural systems, that rely on proteolysis, and can be extended to feature other soluble and membrane proteins, making both systems a powerfull tool for different applications.
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