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KMID : 0545120090190070685
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Journal of Microbiology and Biotechnology
2009 Volume.19 No. 7 p.685 ~ p.689
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Expression and In Vitro Activity of Recombinant Canstatin in Stably Transformed Bombyx mori Cells
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Lee Ji-Hye
Lee Jong-Min
Jeon Hwang-Bo
Sohn Bong-Hee
Yang Jai-Myung
Chung In-Sik
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Abstract
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We describe the expression of recombinant canstatin from stably transformed Bombyx mori Bm5 (Bm5) cells. Recombinant canstatin was secreted into a culture medium with a molecular mass of approximately 29 kDa. Densitometric scanning showed that the secreted canstatin accounted for approximately 91% of the total canstatin production. Recombinant canstatin was also purified to homogeneity using a simple one-step Ni-NTA affinity fractionation. The identity of the purified protein was confirmed as human canstatin by nano-LC-MS/MS analysis. Purified recombinant canstatin inhibited human endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED50) for recombinant canstatin expressed in stably transformed Bm5 cells was approximately 0.64 mg/ml. A maximum production level of 11 mg/l recombinant canstatin was obtained in a T-flask culture of Bm5 cells after 6 days of incubation.
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KEYWORD
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Recombinant canstatin, Bombyx mori, expression, purification, in -vitro activity
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