Production of recombinant human microplasminogen and pilot study in inducing posterior vitreous detachment.
OBJECTIVE
To realize the high production of recombinant human microplasminogen (r-mPlg) with Pichia pastoris and demonstrate the efficacy of r-mPlg in inducing posterior vitreous detachment (PVD).


METHODS
Recombinant plasmid pAO815-3mPlg was constructed and transformed into SMD1168 cells. Positive recombinant clones were selected with MD plate and cultured in BMG medium, then induced in BMM medium. A protein band corresponding to mPlg with molecular mass of 29 kDa was revealed in SDS-PAGE and confirmed by Western blot. Anion-exchange chromatography and plasminogen activity assay kit were used to obtain purified r-mPlg with biological activity. Twenty eyes of freshly slaughtered pigs were divided into 4 groups, 5 eyes in each group. Group 1 served as normal control. Intravitreal injection of 0.1 ml BSS, 1000 IU/0.1 ml recombinant streptokinase (r-SK) and 1000 IU/0.1 ml r-SK plus 3 U/0.1 ml r-mPlg was done respectively to groups 2, 3, and 4. After incubation at 37 degrees C for 60 min, all eyes were processed for light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM).


RESULTS
r-mPlg, which has potential fibrinolytic activity, was successfully obtained with yield of 30 mg/L and purity of 97%. PVD was demonstrated by SEM in group 4 but not in other three groups. The retina and the inner limiting membrane (ILM) were well preserved in all eyes.


CONCLUSIONS
r-mPlg, which has potential fibrinolytic activity, can be produced through Pichia pastoris expression system. Three U of r-mPlg combined with 1000 IU r-SK was effective in producing PVD without damaging the retina.
Qu
Jinfeng
J
People's Hospital of Peking University, Beijing, China.
Li
Xiaoxin
X
Huang
Bingren
B
D002460:Cell Line; D006801:Humans; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000302:isolation & purification; D010843:Pichia / Q000166:cytology; D010865:Pilot Projects; D010958:Plasminogen / Q000096:biosynthesis* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D020255:Vitreous Detachment / Q000209:etiology* / Q000473:pathology
D010446:Peptide Fragments; D011994:Recombinant Proteins; C111879:microplasminogen protein, human; D010958:Plasminogen