Related Experiment Video
Updated: Jul 24, 2026
A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells
Published on: June 10, 2015
High-level synthesis of recombinant murine endostatin in Chinese hamster ovary cells
R M Chura-Chambi1, P H Tornieri, P J Spencer
1Molecular Biology Center, National Nuclear Energy Commission (IPEN-CNEN), Travessa R, 400, Cidade Universitária, 05508-900 São Paulo, Brazil.
Abstract:
Endostatin, a carboxy-terminal fragment of collagen XVIII, has been shown to act as an anti-angiogenic agent that specifically inhibits proliferation of endothelial cells and growth of various primary tumors. Here, we describe the expression by Chinese hamster ovary (CHO) cells of murine endostatin and of a tagged-fusion protein, (his)6-met-endostatin. A dicistronic mRNA expression vector was utilized in which endostatin cDNA was inserted upstream of the amplifiable marker gene, dihydrofolate reductase (DHFR). After transfection of the expression vectors, stepwise increments in methotrexate levels in the culture medium were applied, promoting gene amplification and increasing expression levels of the proteins of interest. The expression level of secreted native endostatin was about 78 microg/mL while the one for secreted (his)6-met-endostatin was about 114 microg/mL, for the best expressing clones. Characterization of physico-chemical and immunological activities of the proteins was performed using SDS-PAGE and Western blotting. The biological activities of recombinant endostatins were tested with a cow pulmonary artery endothelial (C-PAE) cell proliferation assay. Both recombinant endostatin and (his)6-met-endostatin inhibited, in a dose-dependent fashion, growth of C-PAE cells stimulated by basic fibroblast growth factor (bFGF).
Insights
Researchers expressed murine endostatin and a tagged fusion protein in Chinese hamster ovary cells. Both recombinant proteins demonstrated anti-angiogenic properties, inhibiting endothelial cell proliferation in vitro.
Area of Science:
- Biochemistry
- Molecular Biology
- Biotechnology
Background:
- Endostatin, a collagen XVIII fragment, is a known anti-angiogenic agent inhibiting endothelial cell proliferation and tumor growth.
- Recombinant protein expression offers a scalable method for producing therapeutic agents.
Purpose of the Study:
- To describe the expression of murine endostatin and a tagged fusion protein, (his)6-met-endostatin, in Chinese hamster ovary (CHO) cells.
- To characterize the physico-chemical, immunological, and biological activities of the recombinant proteins.
Main Methods:
- Utilized a dicistronic mRNA expression vector with endostatin cDNA and dihydrofolate reductase (DHFR) for gene amplification.
- Employed stepwise methotrexate increments to enhance gene amplification and protein expression levels.
- Analyzed protein expression using SDS-PAGE and Western blotting; assessed biological activity via a C-PAE cell proliferation assay.
Main Results:
- Achieved high expression levels of secreted native endostatin (~78 microg/mL) and (his)6-met-endostatin (~114 microg/mL) in CHO cells.
- Confirmed physico-chemical and immunological properties of the recombinant proteins.
- Demonstrated dose-dependent inhibition of basic fibroblast growth factor (bFGF)-stimulated C-PAE cell proliferation by both recombinant endostatins.
Conclusions:
- Successfully established a system for high-level expression of biologically active murine endostatin and a tagged variant in CHO cells.
- Recombinant endostatins exhibit potent anti-angiogenic activity, validating their potential therapeutic applications.
