Purification of active herpes simplex virus-1 protease expressed in Escherichia coli.
Assembly of viral capsids for replication of herpes simplex virus requires the proteolytic processing of the assembly protein ICP35. The protease responsible for this process is encoded within the 635-amino acid open reading frame of the UL26 gene of the virus. A simple purification scheme is given in this report for the native, mature form of the protease expressed in Escherichia coli. The scheme allows the preparation of milligram quantities of purified enzyme for elucidation of kinetic mechanism as well as for structural studies. Utilizing a 13-residue peptide substrate representing the natural cleavage site that releases the protease, kcat and Km 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 possibility exists that viral or cellular factors are involved in vivo for activation of the protease for herpes capsid maturation.
Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486.
P L
PL
Darke
E
E
Chen
D L
DL
Hall
M K
MK
Sardana
C A
CA
Veloski
R L
RL
LaFemina
J A
JA
Shafer
L C
LC
Kuo
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D012697:Serine Endopeptidases / Q000302:isolation & purification*; D014764:Viral Proteins
D011994:Recombinant Proteins; D014764:Viral Proteins; C079287:scaffold protein, Herpes simplex virus-1; D012697:Serine Endopeptidases