PMID- 8921847
OWN - NLM
STAT- MEDLINE
DCOM- 19961226
LR  - 20190707
IS  - 0378-1119 (Print)
IS  - 0378-1119 (Linking)
VI  - 177
IP  - 1-2
DP  - 1996 Oct 24
TI  - Secretory production of recombinant urokinase-type plasminogen activator-annexin 
      V chimeras in Pichia pastoris.
PG  - 69-76
AB  - 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.
FAU - Okabayashi, K
AU  - Okabayashi K
AD  - Research Division, Green Cross Corporation, Osaka, Japan. okaken@greencross.co.jp
FAU - Tsujikawa, M
AU  - Tsujikawa M
FAU - Morita, M
AU  - Morita M
FAU - Einaga, K
AU  - Einaga K
FAU - Tanaka, K
AU  - Tanaka K
FAU - Tanabe, T
AU  - Tanabe T
FAU - Yamanouchi, K
AU  - Yamanouchi K
FAU - Hirama, M
AU  - Hirama M
FAU - Tait, J F
AU  - Tait JF
FAU - Fujikawa, K
AU  - Fujikawa K
LA  - eng
PT  - Journal Article
PL  - Netherlands
TA  - Gene
JT  - Gene
JID - 7706761
RN  - 0 (Annexin A5)
RN  - 0 (DNA, Recombinant)
RN  - 0 (Recombinant Fusion Proteins)
RN  - EC 3.4.21.73 (Urokinase-Type Plasminogen Activator)
SB  - IM
MH  - Amino Acid Sequence
MH  - Annexin A5/*genetics/isolation & purification/metabolism
MH  - Base Sequence
MH  - Cell Membrane/metabolism
MH  - DNA, Recombinant
MH  - Fibrinolysis
MH  - Genetic Vectors
MH  - Molecular Sequence Data
MH  - Mucor
MH  - Pichia/*genetics
MH  - Recombinant Fusion Proteins/genetics/isolation & purification/metabolism
MH  - Transformation, Genetic
MH  - Urokinase-Type Plasminogen Activator/*genetics/isolation & 
      purification/metabolism
EDAT- 1996/10/24 00:00
MHDA- 1996/10/24 00:01
CRDT- 1996/10/24 00:00
PHST- 1996/10/24 00:00 [pubmed]
PHST- 1996/10/24 00:01 [medline]
PHST- 1996/10/24 00:00 [entrez]
AID - 0378-1119(96)00272-7 [pii]
AID - 10.1016/0378-1119(96)00272-7 [doi]
PST - ppublish
SO  - Gene. 1996 Oct 24;177(1-2):69-76. doi: 10.1016/0378-1119(96)00272-7.