Processing of DNase Domain during Translocation of Colicin E7 across the Membrane of Escherichia coli

• Main Authors: Chen-Chung Liao, 廖辰中
• Other Authors: Kin-Fu Chak
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/08635818431416921540

博士 === 國立陽明大學 === 生物化學研究所 === 89 === Translocation of colicin across the membrane of sensitive cells has been studied extensively. Processing of the toxicity domain of colicin during translocation has been the subject of much controversy. To investigate the final translocation product of colicin across the membrane of E. coli, an expression vector containing His-tagged-cei gene was constructed and introduced into E. coli host cells. The endogenous expressed His-tagged Im7 protein was then used to detect any translocation product containing the DNase domain traversed the inner membrane into cytoplasm of the E. coli cells. As a result, a final processed DNase domain of ColE7 was located in the intracellular space of the cells treated with Col-Im complex. The results of time course experiment indicated that incubation time of cells treated with Col-Im was the function of the amount increased of the processed DNase domain of ColE7 during translocation across the membrane of E. coli. To elucidate the fate of colicin in periplasmic space in vitro, periplasmic extracts were mixed and incubated with purified ColE7. The processed products were also observed. These results suggested that processing of ColE7 is occurred for translocation of the DNase-type colicin across the membrane and the process may probably be taken place in the periplasmic space of the membrane. The binding assay of T2A domain and Im protein by stopped flow and BIACO in acidic condition demonstrate that the low pH condition in periplasmic space might play a role for the dissociation of toxicity domain and immunity protein. In addition, in the presence of periplasmic extracts the process of DNase domain and translocation of the DNase domain across the inner membrane was also observed. Thus, a possible mechanism for colicin is proposed. After the binding of ColE7-Im complex to its target cell, toxicity domain in the C terminal of colicin will be cleaved by certain periplasmic proteins leading to the dissociation of Im protein with the toxicity domain in periplasmic space. Moreover, the low pH condition in periplasmic space might play a role for the dissociation of toxicity domain and immunity protein. Eventually, the processed toxicity domain translocates into cytoplasm through the inner membrane by periplasm proteins to launch its cytotoxic effect in sensitive cells.