Azam Rahimpour1, Roshanak Ahani2, Azita Najaei3, Ahmad Adeli4, Farzaneh Barkhordari4, Fereidoun Mahboudi4. 1. Department of Tissue engineering, Faculty of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, 1985717443, Iran. 2. Department of Virology, Pasteur Institute of Iran, Tehran, 1316943551, Iran. 3. Department of Biochemistry, Payame Noor University, Tehran, 1659639884, Iran. 4. Biotechnology Research Center, Pasteur Institute of Iran, Tehran, 1316943551, Iran.
Abstract
BACKGROUND: Chinese hamster ovary (CHO) cells are the most commonly used host system for the expression of high quality recombinant proteins. However, the development of stable, high-yielding CHO cell lines is a major bottleneck in the industrial manufacturing of therapeutic proteins. Therefore, different strategies such as the generation of more efficient expression vectors and establishment of genetically engineered host cells have been employed to increase the efficiency of cell line development. In order to examine the possibility of generating improved CHO host cells, cell line engineering approaches were developed based on ceramide transfer protein (CERT), and X-box binding protein 1s (XBP1s). METHODS: CHO cells were transfected with CERT S132A, a mutant variant of CERT which is resistant to phosphorylation, or XBP1s expression plasmids, and then stable cell pools were generated. Transient expression of t-PA was examined in engineered cell pools in comparison to un-modified CHO host cells. RESULTS: Overexpression of CERT S132A led to the enhancement of recombinant tissue plasminogen activator (t-PA) expression in transient expression by 50%. On the other hand, it was observed that the ectopic expression of the XBP1s, did not improve the t-PA expression level. CONCLUSION: The results obtained in this study indicate successful development of the improved CHO host cells through CERT S132A overexpression.
BACKGROUND:Chinese hamster ovary (CHO) cells are the most commonly used host system for the expression of high quality recombinant proteins. However, the development of stable, high-yielding CHO cell lines is a major bottleneck in the industrial manufacturing of therapeutic proteins. Therefore, different strategies such as the generation of more efficient expression vectors and establishment of genetically engineered host cells have been employed to increase the efficiency of cell line development. In order to examine the possibility of generating improved CHO host cells, cell line engineering approaches were developed based on ceramide transfer protein (CERT), and X-box binding protein 1s (XBP1s). METHODS:CHO cells were transfected with CERTS132A, a mutant variant of CERT which is resistant to phosphorylation, or XBP1s expression plasmids, and then stable cell pools were generated. Transient expression of t-PA was examined in engineered cell pools in comparison to un-modified CHO host cells. RESULTS: Overexpression of CERTS132A led to the enhancement of recombinant tissue plasminogen activator (t-PA) expression in transient expression by 50%. On the other hand, it was observed that the ectopic expression of the XBP1s, did not improve the t-PA expression level. CONCLUSION: The results obtained in this study indicate successful development of the improved CHO host cells through CERTS132A overexpression.
Entities:
Keywords:
CHO cells; cell engineering; tissue plasminogen activator