Cloning and Expression of Truncated Protein of Epidermal Growth Factor-1 (EGFR-1) in Pichia Pastoris Yeast Host
Background: Epidermal growth factor receptor (EGFR) plays a major role in the pathophysiology of a wide variety of solid tumors such as glioblastoma and breast cancer. Therefore, blocking of signaling cascade of this receptor via specific antibodies is an appropriate therapeutic target against these...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | fas |
| Published: |
Isfahan University of Medical Sciences
2016-02-01
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| Series: | مجله دانشکده پزشکی اصفهان |
| Subjects: | |
| Online Access: | http://jims.mui.ac.ir/index.php/jims/article/view/5306 |
| Summary: | Background: Epidermal growth factor receptor (EGFR) plays a major role in the pathophysiology of a wide variety of solid tumors such as glioblastoma and breast cancer. Therefore, blocking of signaling cascade of this receptor via specific antibodies is an appropriate therapeutic target against these cancers. The first step to make monoclonal antibodies is production of recombinant protein with high purity and glycosylation pattern similar to human protein. One of the best available hosts for this purpose is the Pichia pastoris yeast.
Methods: Coding sequence of extracellular and transmembrane domain of EGFR protein was isolated from human glioma cell line (A172) using real-time polymerase chain reaction (RT-PCR) technique. This sequence was cloned into plasmid pPicZαA and transferred into Pichia pastoris yeast cells. Then, the production of recombinant protein was induced via treating of cells with methanol with final concentration of 0.5%, in several time periods, 24, 48, 72, 96, 120, 144, 168 and 192 hours. Protein production was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Findings: Partial coding sequence of EGFR was cloned in pICZαA plasmid. The results of induction of protein expression on SDS-PAGE gel showed that the protein expression increased as incubation time increased. However, after three days of induction, the secreted host proteins in the culture medium increased and got visible on SDS-PAGE gel.
Conclusion: In this study, we produced EGFR protein that can dramatically speed up production process of EGFR inhibiting monoclonal antibodies. |
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| ISSN: | 1027-7595 1735-854X |