Production in eukaryotic cells and characterization of four hybrids of tissue-type and urokinase-type plasminogen activators

DNA
L PiérardA Bollen

Abstract

To investigate the structure-function relationship in tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), four hybrid sequences were amplified and overexpressed in a mouse myeloma cell line. The following constructs were made starting from cDNA encoding human t-PA and u-PA: (i) a hybrid in which amino acids (AA) 1-262 of the A-chain of t-PA is fused to AA 139-411 of the B-chain of u-PA; (ii) a hybrid in which the kringle 2 region of t-PA (AA 173-262) is inserted between amino acids 130 and 139 of u-PA; (iii) hybrid #2 having amino acids 1 to 10 deleted and replaced by the finger region of t-PA (AA 1-50); and (iv) a chimera in which the finger region of t-PA is followed by amino acids 10-411 of u-PA and where the lysine residues at positions 135 and 136 of u-PA are replaced by glutamines. These four hybrids were efficiently secreted into the culture medium as single-chain polypeptides of the expected molecular weights and had fully functional catalytic activity. Replacement of the A-chain of u-PA by that of t-PA leads to increased fibrin binding, whereas additions of finger and kringle domains do not. These data suggest that structural domains in serine proteases may not fold and/or function...Continue Reading

References

Dec 1, 1977·Proceedings of the National Academy of Sciences of the United States of America·F SangerA R Coulson
Sep 1, 1979·Proceedings of the National Academy of Sciences of the United States of America·H TowbinJ Gordon
Feb 9, 1978·Nature·W Gilbert
Jul 1, 1986·The Journal of General Virology·C R Howard
Jul 1, 1986·Proceedings of the National Academy of Sciences of the United States of America·A J van ZonneveldH Pannekoek
Sep 1, 1984·Proceedings of the National Academy of Sciences of the United States of America·T NyB Lund
Apr 1, 1983·Bioscience Reports·P HérionA Bollen
Apr 27, 1984·Science·H Neurath

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