Specific and efficient cleavage of fusion proteins by recombinant plum pox virus NIa protease

• Published in: Protein expression and purification 2008-02, Vol.57 (2), p.153-162
• Main Authors: Zheng, Nuoyan, Pérez, José de Jesús, Zhang, Zhonghui, Domínguez, Elvira, Garcia, Juan Antonio, Xie, Qi
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Capsid Proteins - isolation & purification; Capsid Proteins - metabolism; Endopeptidases - chemistry; Endopeptidases - isolation & purification; Endopeptidases - metabolism; Enzyme Stability - drug effects; Escherichia coli; Escherichia coli - metabolism; Fusion protein; Fusion tag cleavage; Green Fluorescent Proteins - isolation & purification; Green Fluorescent Proteins - metabolism; Lycopersicon esculentum; Molecular Sequence Data; Nia; Osmolar Concentration; Plum pox virus; Plum Pox Virus - enzymology; Potyviridae; PPV NIa protease; Protease Inhibitors - pharmacology; Protein Processing, Post-Translational - drug effects; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; SARS coronavirus; Sequence Alignment; Sodium Chloride - pharmacology; Substrate Specificity - drug effects; Temperature; Tobacco etch virus; Viral Proteins - chemistry; Viral Proteins - isolation & purification; Viral Proteins - metabolism
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2007.10.008

Site-specific proteases are the most popular kind of enzymes for removing the fusion tags from fused target proteins. Nuclear inclusion protein a (NIa) proteases obtained from the family Potyviridae have become promising due to their high activities and stringencies of sequences recognition. NIa proteases from tobacco etch virus (TEV) and tomato vein mottling virus (TVMV) have been shown to process recombinant proteins successfully in vitro. In this report, recombinant PPV (plum pox virus) NIa protease was employed to process fusion proteins with artificial cleavage site in vitro. Characteristics such as catalytic ability and affecting factors (salt, temperature, protease inhibitors, detergents, and denaturing reagents) were investigated. Recombinant PPV NIa protease expressed and purified from Escherichia coli demonstrated efficient and specific processing of recombinant GFP and SARS-CoV nucleocapsid protein, with site F (N V V V H Q▾A) for PPV NIa protease artificially inserted between the fusion tags and the target proteins. Its catalytic capability is similar to those of TVMV and TEV NIa protease. Recombinant PPV NIa protease reached its maximal proteolytic activity at approximately 30°C. Salt concentration and only one of the tested protease inhibitors had minor influences on the proteolytic activity of PPV NIa protease. Recombinant PPV NIa protease was resistant to self-lysis for at least five days.