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

A gene encoding the human rhinovirus 14 (HRV14) sequence for expressio n of the viral polypeptide protein Delta 3ABC was inserted into a plas mid driven by the heat-inducible bacteriophage lambda P-L promoter. Th e coding sequence was also inserted into a PET vector for expression i n the T7 system to produce C-13, N-15-labeled protein. The expressed H RV14 3C protease (3C(pro)) autocatalytically cleaved itself from the p olyprotein Delta 3ABC, and the mature HRV14 3C(pro) partitioned predom inantly, in the case of the T7 system, in the insoluble fraction and e xclusively, 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 an thranilic acid (Anc)-Thr-Leu-Phe-Gln-Gly-Pro-Val-(p-NO2)-Phe-Lys mimic ked the natural 2C/3A cleavage site (Thr-Leu-Phe-Gln-Gly-Pro-Val-Tyr-P he) using an N-terminal anthranilic acid donor group on one side of th e scissile bond (Gln/Gly) and a p-NO2-Phe acceptor group at the P4 pos ition. Measured by the fluorescence assay, HRV14 3C(pro) had a K-m of 300 mu M for the substrate. (C) 1995 Academic Press, Inc.