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Production of recombinant human microplasminogen and pilot study in inducing posterior vitreous detachment
Jinfeng Qu1, Xiaoxin Li, Bingren Huang
1People's Hospital of Peking University, Beijing, China.
Purpose:
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.
Conclusion:
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.
Insights
Recombinant human microplasminogen (r-mPlg) was successfully produced and demonstrated to induce posterior vitreous detachment (PVD) safely. This fibrinolytic agent shows promise for ophthalmic applications without damaging retinal tissues.
Area of Science:
- Biotechnology
- Ophthalmology
- Protein Engineering
Background:
- Posterior vitreous detachment (PVD) is a common age-related condition.
- Current treatments for PVD are limited, necessitating novel therapeutic approaches.
- Recombinant protein expression offers a pathway for developing new ophthalmic agents.
Purpose of the Study:
- To achieve high-level production of recombinant human microplasminogen (r-mPlg) using the Pichia pastoris expression system.
- To evaluate the efficacy of r-mPlg in inducing posterior vitreous detachment (PVD) in a preclinical model.
- To assess the safety of r-mPlg in preserving retinal structure and function.
Main Methods:
- Construction and transformation of the pAO815-3mPlg plasmid into Pichia pastoris.
- Culturing and induction of recombinant protein expression, followed by SDS-PAGE and Western blot validation.
- Purification of r-mPlg using anion-exchange chromatography and assessment of biological activity.
- Intravitreal injection in porcine eyes followed by histological and electron microscopy analysis.
Main Results:
- High-yield (30 mg/L) and high-purity (97%) r-mPlg with potential fibrinolytic activity was successfully obtained.
- Scanning electron microscopy confirmed PVD induction in eyes treated with r-mPlg and recombinant streptokinase (r-SK).
- No damage to the retina or inner limiting membrane (ILM) was observed in any treatment group.
Conclusions:
- The Pichia pastoris system is effective for producing functional r-mPlg.
- A combination of r-mPlg and r-SK can induce PVD effectively.
- r-mPlg demonstrates a favorable safety profile for potential use in ophthalmic procedures.
