Author/Authors :
Majidzadeh، Keivan نويسنده Tasnim Biotechnology Research Center, Tehran, Iran , , Mahboudi، Fereidoun نويسنده Soleimani, M , Hemayatkar، Mahdi نويسنده Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran , , Davami، Fatemeh نويسنده Electrical and Computer Engineering Department Davami, Fatemeh , Barkhordary، Farzaneh نويسنده Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran , , Adeli، Ahmad نويسنده Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran , , Soleimani، Mohammad نويسنده Davudi, N , Davoudi، Noushin نويسنده Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran , , Khalaj، Vahid نويسنده Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran ,
Abstract :
Tissue plasminogen activator (tPA) is a serine protease, which is composed of five distinct structural domains with 17 disulfide bonds, representing a model of high-disulfide proteins in human body. One of the most important limitations for high yield heterologous protein production in Escherichia coli
(E. coli) is the expression of complex proteins with multiple disulfide bridges. In this study the combination of two distinct strategies, manipulated cytoplasm and native periplasm, was applied to produce the functional full length tPA enzyme in E. coli. Using a PelB signal peptide sequence at 5ʹ site of tPA gene, the expression cassette was prepared and subsequently was transformed into a strain with manipulated oxidizing cytoplasm. Then the induction was made to express the protein of interest. The SDS-PAGE analysis and gelatin hydrolysis confirmed the successful expression of functional tPA. The results of this study showed that complex proteins can be produced in E. coli using the cumulative power of both cytoplasm and periplasm.
