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Efficiency of translation and post-translation regulatory genes in optimization of tissue plasminogen activator gene expression
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Azam Rahimpour   , Azita Najaei , Fereidoun Mahboudi |
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Abstract: (4543 Views) |
| Introduction: Chinese hamster ovary (CHO) cells are the most commonly
used host system for the expression of high quality recombinant proteins.
Different strategies such as generation of more efficient expression vectors
and establishment of genetically engineered host cells have been employed to
improve recombinant protein expression in this system. Here the cell line
engineering strategy based on the phosphorylation resistant variants of
eukaryotic initiation factor 2 alpha(eIF2α) and ceramide transfer protein(CERT),
key mediators of protein translation and secretion, have been used to develop
improved CHO host cells. Materials and Methods: The CERT S132A, peIF2α S51A and tissue plasminogen activator
(t-PA) were cloned in suitable expression vectors. CERT S132A and peIF2α S51A
stable cell pools were generated and the efficacy of the cells for transient
expression of t-PA was evaluated in comparison to un-modified CHO cells. Results: Integration
of CERT S132A and peIF2α S51A expression vectors in CHO cell genome confirmed
by PCR analysis of genomic DNA from stable cells. Moreover, the specific bands
for CERT S132A and peIF2α S51A proteins were observed in Western blot analysis.
Expression of CERT S132A gene increased expression of t-PA by 50%. However no positive
effect on t-PA expression was observed in the peIF2α S51A stable cell pools. Conclusion: This study showed the efficiency of CERT S132Abased cell line
engineering approach for the development of optimized CHO host cells |
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| Keywords: CHO, Eukaryotic initiation factor 2, Ceramides, Tissue plasminogen activator |
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Full-Text [PDF 846 kb]
(1198 Downloads)
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Type of Study: Research |
Subject:
General
Received: 2015/08/10 | Accepted: 2015/09/7 | Published: 2015/09/26
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