[The cloning expression, purification and activity of human angiostatin K (1-3) gene].
OBJECTIVE
To clone the sequence of human Angiostatin cDNA and obtain the protein of recombinant angiostatin for further development.


METHODS
Fresh human liver tissue was used for the extraction of total RNA and amplified the Angiostatin cDNA through RT-PCR method . After the recombinant plasmid pET30a-Angiostatin was constructed and confirmed, it was transduced into Rosetta (DE3). Then the transformation was used for fermentation and induced expression. The protein was identified by Western Blot and purified by Ni-NTA affinity chromatography.


RESULTS
The sequence of human Angiostatin cDNA was identical with genebank. Angiostatin (K1-3) was expressed and purified. The target protein made up 30% of the total bacterial protein. The purity is above 90%.


CONCLUSIONS
Angiostatin K (1-3) can be reached using fusion vector pET30a. The product have the biological activity.
Wang
Yun-Long
YL
Zhengzhou University of Light Industry, Zhengzhou 450002, China.
Wang
Guo-Qiang
GQ
Li
Zhi-Tao
ZT
Lu
Xiao-Ya
XY
Li
Yu-Lin
YL
Wang
Zhen
Z
D043168:Angiostatins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008099:Liver / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D043168:Angiostatins