PROTEIN ENGINEERING IN THE STUDY OF PROTEIN LABELING AND DEGRADATION

Proteins are large macromolecules that play important roles in nature. With the development of modern molecular biology techniques, protein engineering has emerged as a useful tool and found many applications in areas ranging from food industry, environmental protection, to medical and life science. Biomimetic membrane incorporates biological elements, such as proteins, to form membranes that mimic the high specificity and conductance of natural biological membranes. For any application involving the usage of proteins, the first barrier is always the production of proteins with sufficient stability, and the incorporation of proteins into the artificial matrix. This thesis contains two major parts, the first part is focused on the development and testing of method to immobilize active enzymes. The second part is devoted to study the degradation of membrane proteins in E. coli cells. In the immobilization study, Pyrophosphatase (PpaC) was chose as a model enzyme. A dual functional tag consist of histidine and methionine has been developed, in which histidine is used for purification while methionine is metabolically replaced with azidohomoalanine (AHA) for immobilization. We found that the addition of the tag and the incorporation of AHA did not significantly impair the properties of proteins, and the histidine–AHA tag can facilitate protein purification, immobilization, and labeling. This tag is expected to be useful in general for many proteins. Degradation of soluble protein has been well characterized, but the membrane protein degradation process remains elusive. SsrA tag is a well-known recognition sequence for soluble protein degradation, which marks prematurely terminated protein products translated from damaged mRNA. SsrA tagged membrane proteins was found to be substrate of a cytosolic protease complex ClpXP, which mediated complete degradation