PURIFICATION OF RECOMBINANT HUMAN RHINOVIRUS-14-3C PROTEASE EXPRESSED IN ESCHERICHIA-COLI

A gene encoding the human rhinovirus 14 (HRV14) sequence for expression of the viral polypeptide protein Delta 3ABC was inserted into a plasmid driven by the heat-inducible bacteriophage lambda P-L promoter. The coding sequence was also inserted into a PET vector for expression in the T7 system to produce C-13, N-15-labeled protein. The expressed HRV14 3C protease (3C(pro)) autocatalytically cleaved itself from the polyprotein Delta 3ABC, and the mature HRV14 3C(pro) partitioned predominantly, in the case of the T7 system, in the insoluble fraction and exclusively, in the case of the P-L system, in the insoluble fraction, The insoluble HRV14 3C(pro) 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 3C(pro) 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 3C(pro) had a K-m of 300 mu M for the substrate. (C) 1995 Academic Press, Inc.