Purification of Recombinant Human Rhinovirus 14 3C Protease Expressed in Escherichia coli

Birch, G. M.; Black, Thomas D.; Malcolm, S; Lai, Mei‐Huei T.; Zimmerman, R. E.; Jaskunas, S R

doi:10.1006/prep.1995.1080

Cited by 31 publications

(25 citation statements)

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“…The availability of these methods has greatly facilitated our understanding of viral 3C protease, especiallywith respect to its substrate specificity and amino acid preferences.These assaying methods, however, are not adequate for high throughput screening owing to their laborious and time consuming procedures. Recently, Birch et al (1995) reported a continuous fluorescenceassay using anthranilyl peptide substrate to detect HRV-14 3C protease. However, high background activitywas observed with the uncleaved substrate, caused by the insufficient collisional quenching by p-nitrophenylalanine.…”

Section: Introductionmentioning

confidence: 99%

Development of a Continuous Fluorescence Assay for Rhinovirus 14 3C Protease Using Synthetic Peptides

Wang

Cohen

et al. 1997

Antivir Chem Chemother

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SummaryRhinovirus 3C protease is an attractive target For therapeutic intervention owing to its important role in virion maturation and inFectivity. In order to Facilitate the identification of potential 3C protease inhibitors, we have developed a continuous Fluorescence assay using 5-[(2-aminoethyl}amino]naphthalene-l-sulphonic acid (Edans) as a Fluorescent donor and 4-(4-dimethylaminophenylazo}benzoic acid (Dabcyl) as a quenching acceptor. Several F1uorogenic peptide substrates For 3C protease were synthesized by both solution chemistry and solid phase peptide synthesis. One of the synthetic Edans/ Dabcyl substrates, with an amino acid sequence derived From the 2C/3A site of the virus polyprotein, yielded a 24-Fold increase in Fluorescence intensity after 3C cleavage. Data regarding substrate cleavage kinetics, assay sensitivity and optimization are presented. The application ofthisassay to the evaluation of 3C protease inhibitors is also shown.

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Section: Introductionmentioning

confidence: 99%

Development of a Continuous Fluorescence Assay for Rhinovirus 14 3C Protease Using Synthetic Peptides

Wang

Cohen

et al. 1997

Antivir Chem Chemother

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“…Previous studies from our group showed that active recombinant HRV14 3C protease could be purified to homogeneity as shown by SDS-polyacrylamide gel electrophoresis and was able to cleave various synthetic peptides (13,14). In this report, we describe the identification of a deamidated isoform of HRV14 3C protease present in the purified enzyme preparation and in the infected cells.…”

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confidence: 72%

“…Protease-HRV14 3C protease was expressed in bacterial cells and purified as described previously (13). The identity of the purified HRV14 3C protease was confirmed by a combination of analyses including N-terminal amino acid sequencing, ion spray mass spectrometry, and SDS-polyacrylamide gel electrophoresis followed by Western blot using polyclonal antibodies raised in rabbits against the purified recombinant HRV14 3C protease.…”

Section: Preparation and Identification Of Recombinant Hrv14 3cmentioning

confidence: 99%

“…The converted pI 8.3 isoform was confirmed by IEF gel analysis of the treated 3C protein sample. Measurement of HRV14 3C Protease Activity-The fluorogenic peptide (aminobenzoic acid)-Thr-Leu-Phe-Gln-Gly-Pro-Val-Phe(p-nitro)-Lys has been shown to be cleaved between glutamine and glycine by HRV14 3C protease (13). The cleavage reaction was performed at 30°C for 60 min in a 400-l mix containing 50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 200 M peptide substrate, and 0.3 M 3C protease.…”

Section: Preparation and Identification Of Recombinant Hrv14 3cmentioning

confidence: 99%

“…To examine the effect of this mutation on viral replication, mutant HRV14 RNA was transfected into HeLa cell monolayers under the conditions described above, and the viral plaque formation was determined. For generation of mutant 3C protein, expression vector carrying the Asp-164 mutation was used to transform competent bacterial cells under the identical conditions used for wild type recombinant 3C protein expression (13).…”

Section: Preparation and Identification Of Recombinant Hrv14 3cmentioning

confidence: 99%

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Identification and Characterization of Human Rhinovirus-14 3C Protease Deamidation Isoform

Cox

Johnson

Cook

et al. 1999

Journal of Biological Chemistry

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A purified recombinant human rhinovirus-14 3C protease preparation contained only ϳ50% active enzyme as titrated using specifically designed irreversible 3C protease inhibitors. Analysis of the purified 3C protein by isoelectric focusing showed differently charged 3C isoforms that had isoelectric points (pI) of 8.3 (55%) and 9.0 (45%), with the latter one being consistent with the predicted pI of the human rhinovirus-14 3C protein. Further analysis indicated that the pI 8.3 protein was the deamidated form of 3C, and it displayed ϳ10-fold reduced cleavage activity relative to the original 3C protease sample. Peptide mapping followed by sequence analysis revealed that a single asparagine, Asn-164, was deamidated to aspartic acid in the pI 8.3 isoform. Converting Asn-164 to Asp by site-directed mutagenesis resulted in a mutated 3C protease with extremely low activity, as seen with the pI 8.3 isoform, indicating a role of Asn-164 in substrate recognition and binding. In addition, the deamidated 3C protease was found to be present in vivo, and its abundance was related to the viral replication cycle. Moreover, mutant virus carrying Asp-164 showed reduced viability in infected cells. Taken together, our data suggest that 3C protein deamidation plays a role in the regulation of its enzymatic activity. Human rhinovirus (HRV)1 infections are considered to be the most frequent causative agents of the common cold and various other upper respiratory tract infections (1). Rhinoviruses are members of the picornavirus family, which also includes the apthoviruses (foot-and-mouth disease virus), cardioviruses (encephalomyocarditis virus), enteroviruses (poliovirus and Coxsackie virus), and hepatitis A virus. All picornaviruses have a positive-sense, single-stranded RNA genome that is translated into a single polyprotein precursor. In the case of HRVs, the viral polyprotein is mainly processed by the viral proteases 2A and 3C to generate functional proteins and enzymes (2, 3). The 2A protease catalyzes the first cleavage between the structural and nonstructural proteins, whereas the 3C protease catalyzes most of the subsequent internal cleavages (2, 3).The availability of active recombinant 3C protease greatly facilitated its biochemical characterization. Purified recombinant viral 3C protease was able to cleave different proteins and peptides at the bond formed between glutamine and glycine (4, 5). It has been found that 3C protease could regulate host cell function by cleaving important cellular proteins during infection (6, 7). In addition to its proteolytic activity, viral 3C protease has been shown to be a RNA-binding protein and may be involved in formation of the viral replication complex (8). As illustrated by its crystal structure, HRV 3C protease represents a novel class of cysteine protease that contains a cysteine as the active site nucleophile but is structurally like a serine protease (9 -11). It has been considered to be an ideal target for antiviral intervention due to its essential role in viral replication and i...

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Scintillation Proximity Assay

Kahl

Felder

1999

CP Neuroscience

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Purification of Recombinant Human Rhinovirus 14 3C Protease Expressed in Escherichia coli

Cited by 31 publications

References 0 publications

Development of a Continuous Fluorescence Assay for Rhinovirus 14 3C Protease Using Synthetic Peptides

Development of a Continuous Fluorescence Assay for Rhinovirus 14 3C Protease Using Synthetic Peptides

Identification and Characterization of Human Rhinovirus-14 3C Protease Deamidation Isoform

Scintillation Proximity Assay

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