Bioactivity and stability analysis of endostatin purified from fermentation supernatant of 293 cells transfected with Ad/rhEndo.
Endostatin is a potent angiogenic inhibitor that has been approved for the treatment of cancer. However, endostatin is unstable in vitro and difficult to produce in large quantities. Endostatin gene therapy is an alternative to overcome these difficulties by expressing sustained bioactive endostatin in vivo. We previously developed a recombinant replication-defective adenovirus, E10A, which carries the human endostatin gene. Phase I trial of E10A given as a weekly intratumoral injection in adult patients with solid tumors has been finished. The clinical application of endostatin gene therapy was limited by the high cost of large-scale production. In the current study, we found that there was a high level (100mg/L) of endostatin in the fermentation supernatant of 293 cells transfected with Ad/rhEndo. A protocol was developed to purify recombinant endostatin in the fermentation supernatant to a yield of 24mg/L and 98% purity by the use of SP Sepharose FF cation exchange chromatography, Sepharose-heparin Hi Trap affinity chromatography and gel filtration chromatography. The anti-proliferative activity of 293 cell-expressed endostatin (ArhEndo) is comparable to that of yeast-expressed endostatin (YrhEndo). Cell migration assay indicated that ArhEndo is more effective than YrhEndo. Moreover, ArhEndo is of higher stability than YrhEndo. These results suggested that purification of recombinant endostatin from fermentation supernatant provided an economic and available strategy for Ad/rhEndo production.
Liang
Zhihui
Z
State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-sen University, Guangzhou, PR China.
Wu
Jiangxue
J
Huang
Jialing
J
Tan
Weiping
W
Ke
Miaola
M
Liu
Ranyi
R
Huang
Bijun
B
Xiao
Xia
X
Zhao
Peng
P
Huang
Wenlin
W
D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D054443:Cell Migration Assays; D049109:Cell Proliferation; D002478:Cells, Cultured; D043169:Endostatins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D005285:Fermentation; D005822:Genetic Vectors; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D043169:Endostatins; D011994:Recombinant Proteins