Therapeutic applications of synthetic biology are providing new treatments for various diseases using innovative principles. These are termed living drugs that can sense a specific input signal and respond to it with synthesis of an effector molecule or drug. Living drugs are complex biological systems that must be tightly controlled. Control over biological systems is achieved with synthetic biology tools that are mostly focused on regulation of gene expression. The most important drawback of this type of regulation is a delay in signal transduction from input to output which is very important in some applications where fast response is required. In thesis we decided to prepare and test a new and innovative system for rapid protein release from post-translationaly controlled cells with proteolysis. Through mimicking natural mechanisms for protein retention in the lumen or at membrane of the endoplasmic reticulum (ER) we delayed protein release by fusion with the ER retention sequence. This was demonstrated by confocal microscopy and Western transfer. Split protease TEVp geneticaly fused to chemically induced dimerization domains was controlled by the addition of a small molecule and used for cleavage off of the ER retention sequence. After reconstitution of the split TEVp protease, retention sequence was cleaved off the protein that resided in the lumen or at the ER membrane causing release of the reporter protein SEAP from cells. SEAP activity was experimentaly determined in cells medium. With all designed systems controlled at the post-translational level, faster release of the protein was achived in comparison to transcriptionally controlled cells. The fastest protein release was achived in the system comprising protein retention in the lumen of the ER and reconstitution of split protease erTEVp whose activity in split form was showed for the first time in the system for the rapid protein release.
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