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

• Published in: Protein expression and purification 1995-10, Vol.6 (5), p.609-618
• Main Authors: Birch, G.M., Black, T., Malcolm, S.K., Lai, M.T., Zimmerman, R.E., Jaskunas, S.R.
• Format: Article
• Language: English
• Subjects: 3C Viral Proteases; Amino Acid Sequence; Base Sequence; Chromatography - methods; Chromatography, High Pressure Liquid; Cysteine Endopeptidases - biosynthesis; Cysteine Endopeptidases - genetics; Cysteine Endopeptidases - isolation & purification; Cytoplasmic Granules - chemistry; Cytoplasmic Granules - metabolism; Escherichia coli - genetics; Fluorescence; Humans; Kinetics; Molecular Sequence Data; Plasmids - chemistry; Plasmids - genetics; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Rhinovirus - enzymology; Solubility; Substrate Specificity; Urea - chemistry; Viral Proteins
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.1995.1080

A gene encoding the human rhinovirus 14 (HRV14) sequence for expression of the viral polypeptide protein Δ3ABC was inserted into a plasmid driven by the heat-inducible bacteriophage λPL promoter. The coding sequence was also inserted into a PET vector for expression in the T7 system to produce 13C, 15N-labeled protein. The expressed HRV14 3C protease (3Cpro) autocatalytically cleaved itself from the polyprotein Δ3ABC, and the mature HRV14 3Cpro partitioned predominantly, in the case of the T7 system, in the insoluble fraction and exclusively, in the case of the PL system, in the insoluble fraction, The insoluble HRV14 3Cpro was solubilized in urea and purified using anion- and cation-exchange chromatography. The protease was refolded/activated and further purified using a size-exclusion column. HRV14 3Cpro was purified to >90% homogeneity as shown by SDS-PAGE and to 95% by HPLC. A continuous fluorescence assay was developed which utilized an intramolecularly quenched 9-amino-acid substrate, The substrate anthranilic acid (Anc)-Thr-Leu-Phe-Gln-Gly-Pro-Val-(p-NO2)-Phe-Lys mimicked the natural 2C/3A cleavage site (Thr-Leu-Phe-Gln-Gly-Pro-Val-Tyr-Phe) using an N-terminal anthranilic acid donor group on one side of the scissile bond (Gln/Gly) and a p-NO2-Phe acceptor group at the P4 position. Measured by the fluorescence assay, HRV14 3Cpro had a Km of 300 μM for the substrate.