Secretory production of recombinant urokinase-type plasminogen activator-annexin V chimeras in Pichia pastoris.
To produce a thrombi-targeting plasminogen activator, we expressed a fused gene that contains a modified pre-sequence of Mucor pussilus rennin (MPR) followed by a chimeric gene of single-chain urokinase-type plasminogen activator (scu-PA)::annexin V (AV). The fused gene was ligated into an integrative vector, under the control of the alcohol oxidase 1 (AOX1) promoter (p), and transformed into Pichia pastoris. Transformants were monitored for the secretion of fibrinolytic activity. The highest expressing clone, HB225, secreted as much as 600 international units (IU) of fibrinolytic activity per ml of culture medium under optimal conditions. It contained three tandem copies of the full-size vector disruptively integrated into the AOX1 sequence. Western blot analysis revealed that the secreted chimera was highly susceptible to proteolysis. Addition of excess amino acids (aa) to the culture medium minimized the degree of proteolysis. Two major species of chimera, 85 and 65 kDa, were then isolated from the culture medium. The former was the intact form consisting of a single-chain and showing full enzyme activity after activation by plasmin. The latter was an enzymatically processed form consisting of two chains held by a disulfide bond, having full enzyme activity without activation. Both chimeras exhibited calcium-dependent phospholipid (PL)-binding affinities similar to the parent AV.
Okabayashi
K
K
Research Division, Green Cross Corporation, Osaka, Japan. okaken@greencross.co.jp
Tsujikawa
M
M
Morita
M
M
Einaga
K
K
Tanaka
K
K
Tanabe
T
T
Yamanouchi
K
K
Hirama
M
M
Tait
J F
JF
Fujikawa
K
K
D000595:Amino Acid Sequence; D017304:Annexin A5 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D004274:DNA, Recombinant; D005342:Fibrinolysis; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009089:Mucor; D010843:Pichia / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014170:Transformation, Genetic; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D017304:Annexin A5; D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D014568:Urokinase-Type Plasminogen Activator