Transformed Lepidopteran insect cells: new sources of recombinant human tissue plasminogen activator.
Stable transformation was used to generate a cloned insect cell line (Bm5 silkmoth cells) over-expressing human tissue plasminogen activator (tPA). This cell line expressed 135 microg/mL single chain tPA in serum-free medium in static culture with a maximum specific activity of 120 IU/microg. In serum-containing medium, this line expressed 160 microg/mL of combined single-chain tPA, two-chain tPA, and a higher molecular weight SDS-stable tPA complex in suspension cultures with a maximum specific activity of 255 IU/microg. Approximately 100 copies of the tPA cDNA were randomly integrated into each Bm5 cell. For secretion of recombinant tPA from Bm5 cells, the native human tPA signal peptide is as effectively recognized as an insect specific signal peptide derived from a silkmoth chorion gene. Finally, stably transformed polyclonal populations of Bm5, High Five, and Sf21 cells expressing tPA were generated and compared for relative tPA expression.
Departments of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
P J
PJ
Farrell
L A
LA
Behie
K
K
Iatrou
D000818:Animals; D001769:Blood; D012831:Bombyx / Q000166:cytology / Q000235:genetics*; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D016895:Culture Media, Serum-Free; D006801:Humans; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000235:genetics*; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D016895:Culture Media, Serum-Free; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator