[Expression and characterization of t-PA in insect cells].
With the RT-PCR technique, human tissue-type plasminogen activator (t-PA) cDNA was amplified from human melanoma cells. The sequence was proved to be the same as those reported by foreign researchers. The t-PA cDNA containing whole reading frame was inserted into the transfer vector pBacPAK8. The constructed pBac-tPA and the linear BacPAK6 virus DNA were cotransfected with Tn-5B-1 insect cells by lipofectin-mediated transfection method. Eleven pure recombinant viruses were isolated by plaque assay, and one of them, nominated BactPA3, was selected by PCR identification and biological activity comparison. The t-PA activity expressed in serum-containing media reach the highest level at 72 h postinfection. The activity was 3.04 x 10(3) IU/ml, e.g. 1.8 x 10(4) IU/10(6) cells. The highest expression level in serum-free media was almost the same, but needed more time (at 132 h postinfection). SDS-PAGE fibrin autography showed that the molecular weight of the expressed t-PA was about 68 kDa. Its stimulation by fibrinogen, affinity with fibrin and inactivation in plasma were almost the same as the native t-PA purified from human melanoma cell culture. The half-life of t-PA expressed in serum-free media was seven minutes.
Liu
J B
JB
Biotechnology Research Center, Zhongshan University, Guangzhou 510275.
Yang
K
K
Xue
A Q
AQ
Pang
Y
Y
Li
B J
BJ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D007313:Insecta / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D018076:DNA, Complementary; D010959:Tissue Plasminogen Activator