Specific and efficient cleavage of fusion proteins by recombinant plum pox virus NIa protease.
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 black triangle down 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 degrees 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.
Zheng
Nuoyan
N
State Key Laboratory for Biocontrol, Sun Yat-sen (Zhongshan) University, 135 Xinggang Road W, Guangzhou 510275, China.
Pérez
José de Jesús
Jde J
Zhang
Zhonghui
Z
Domínguez
Elvira
E
Garcia
Juan Antonio
JA
Xie
Qi
Q
D000595:Amino Acid Sequence; D036022:Capsid Proteins / Q000302:isolation & purification / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D017801:Plum Pox Virus / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature; D014764:Viral Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D036022:Capsid Proteins; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D012965:Sodium Chloride; D010450:Endopeptidases; C059992:nuclear inclusion protein a, mosaic viruses