PURIFICATION OF ACTIVE HERPES-SIMPLEX VIRUS-1 PROTEASE EXPRESSED IN ESCHERICHIA-COLI

Assembly of viral capsids for replication of herpes simplex virus requ ires the proteolytic processing of the assembly protein ICP35. The pro tease responsible for this process is encoded within the 635-amino aci d open reading frame of the U(L)26 gene of the virus. A simple purific ation scheme is given in this report for the native, mature form of th e protease expressed in Escherichia coil. The scheme allows the prepar ation of milligram quantities of purified enzyme for elucidation of ki netic mechanism as well as for structural studies. Utilizing a 13-resi due peptide substrate representing the natural cleavage site that rele ases the protease, k(cat) and K-m values of the purified native enzyme are 2.0 min(-1) and 0.88 mM, respectively. Thus, peptide cleavage is less efficient than reported for other viral proteases. The possibilit y exists that viral or cellular factors are involved in vivo for activ ation of the protease for herpes capsid maturation.