Human tissue-type plasminogen activator synthesized by using a baculovirus vector in insect cells compared with human plasminogen activator produced in mouse cells.
A cDNA fragment encoding the human tissue-type plasminogen activator was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus downstream from the polyhedrin promoter. The induction kinetics of t-PA was followed, after infection of Spodoptera frugiperda cells, at both mRNA and protein levels. Fibrinolytically active plasminogen activator accumulated in the culture medium and reached 2.5 micrograms/ml after 120 h. The protein was compared with recombinant plasminogen activator produced in mouse cells and was found to be slightly smaller. This difference in size was found to be caused by N-linked oligosaccharides which are shorter in the recombinant activator obtained from insect cells. The molecules produced in such cells contain at least two different types of N-linked glycans, since only one out of three oligosaccharides is sensitive to endoglycosidase H. However, all glycan structures bind strongly to concanavalin A-Sepharose.
Steiner
H
H
Department of Microbiology, University of Stockholm, Sweden.
Pohl
G
G
Gunne
H
H
Hellers
M
M
Elhammer
A
A
Hansson
L
L
D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000378:metabolism; D006801:Humans; D007304:Insect Viruses / Q000235:genetics*; D007700:Kinetics; D051379:Mice; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator