Single amino acid substitutions in recombinant plant-derived human 伪₁-proteinase inhibitor confer enhanced stability and functional efficacy

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• 作者：Shweta Jha ; Indraneel Sanyal ; D.V. Amla
• 关键词：伪1-PI ; 伪1-proteinase inhibitor ; CD ; circular dichroism ; Cm ; mid-point of transition ; DAC-ELISA ; direct antigen coating-enzyme linked immunosorbent assay ; PPE ; porcine pancreatic elastase ; RCL ; reactive center loop ; TSP ; total soluble protein ; TUG ; transverse urea gradient gel electrophoresis
• 刊名：Biochimica et Biophysica Acta (BBA)/General Subjects
• 出版年：January, 2014
• 年：2014
• 卷：1840
• 期：1
• 页码：416-427
• 全文大小：1233 K

文摘

Background

Human 伪₁-proteinase inhibitor (伪₁-PI) is the most abundant serine protease inhibitor in the blood and the heterologous expression of recombinant 伪₁-PI has great potential for possible therapeutic applications. However, stability and functional efficacy of the recombinant protein expressed in alternate hosts are of major concern.

Methods

Five variants of plant-expressed recombinant 伪₁-PI protein were developed by incorporating single amino acid substitutions at specific sites, namely F51C, F51L, A70G, M358V and M374I. Purified recombinant 伪₁-PI variants were analyzed for their expression, biological activity, oxidation-resistance, conformational and thermal stability by DAC-ELISA, porcine pancreatic elastase (PPE) inhibition assays, transverse urea gradient (TUG) gel electrophoresis, fluorescence spectroscopy and far-UV CD spectroscopy.

Results

Urea-induced unfolding of recombinant 伪₁-PI variants revealed that the F51C mutation shifted the mid-point of transition from 1.4 M to 4.3 M, thus increasing the conformational stability close to the human plasma form, followed by F51L, A70G and M374I variants. The variants also exhibited enhanced stability for heat denaturation, and the size-reducing substitution at Phe51 slowed down the deactivation rate ~ 5-fold at 54 掳C. The M358V mutation at the active site of the protein did not significantly affect the conformational or thermal stability of the recombinant 伪₁-PI but provided enhanced resistance to oxidative inactivation.

Conclusions

Our results suggest that single amino acid substitutions resulted in improved stability and oxidation-resistance of the plant-derived recombinant 伪₁-PI protein, without inflicting the inhibitory activity of the protein.

General significance

Our results demonstrate the significance of engineered modifications in plant-derived recombinant 伪₁-PI protein molecule for further therapeutic development.