Expression and characterization of recombinant human micro-plasminogen.
Micro-plasminogen (microPlg) gene amplified from human liver cells by reverse transcription PCR was inserted into expression vector pET-28a (pET-28a/microPlg) and transformed into E. coli strain BL21(DE3). Recombinant human micro-plasminogen (rh-microPlg) was over-expressed as inclusion bodies when induced with IPTG. After renaturation and purification, 16 mg rh-microPlg/l was obtained with a homogeneity of 95% (w/w). Pro-urokinase (proUK)-induced rh-microPlg activation was significantly faster than when Glu-plasminogen was the substrate. The catalytic efficiency of urokinase (UK) activation of rh-microPlg was twice that of Glu-plasminogen. While recombinant human micro-plasmin (rh-microPlm) and Lys-plasmin had a similar amidolytic activity against a small substrate, D-valyl-L-leucyllysine-p-nitroaniline dihydrochloride, Lys-plasmin activated proUK with a catalytic efficiency about fourfold greater than did rh-microPlm. These results suggested that the kringle 1-5 domain of plasminogen and plasmin may modify both UK activation of plasminogen and plasmin activation of proUK, respectively.
Ma
Zhifeng
Z
Institute of Molecular Medicine, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, China.
Lu
Wei
W
Wu
Sheng
S
Chen
Junyong
J
Sun
Ziyong
Z
Liu
Jian-Ning
JN
D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010958:Plasminogen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry*
D010446:Peptide Fragments; D011994:Recombinant Proteins; C111879:microplasminogen protein, human; D010958:Plasminogen; D014568:Urokinase-Type Plasminogen Activator