Expression of natural and synthetic genes encoding herpes simplex virus 1 protease in Escherichia coli and purification of the protein.
An attractive target for anti-herpes chemotherapy is the herpes simplex virus 1 (HSV-1) protease encoded by the UL26 gene. Studies with HSV-1 strains that harbour mutations in the protease gene have demonstrated that the protease is essential for DNA packaging and virus maturation. The UL26 translation product is 635 amino acids long and undergoes autoproteolytic processing between residues Ala247/Ser248 and Ala610/Ser611. The N-terminal processing product (amino acids 1-247) contains the protease domain. To perform crystallization studies and high throughput screening for potent inhibitors, large amounts of the HSV-1 protease are required. However, expression of the natural HSV-1 protease gene in Escherichia coli using a T7-promoter-regulated system is low and does not allow for the efficient production of larger amounts of highly purified enzyme. In this report, we describe the use of a synthetic protease gene with optimized E. coli codon usage. The level of protease expression was at least 20 times higher with the synthetic gene as compared to the natural UL26 gene. The HSV-1 protease was purified to homogeneity in three steps using mixed-bed ion-exchange chromatography, affinity chromatography, and hydroxyapatite chromatography.
Apeler
H
H
Pharma Research, Bayer AG, Wuppertal, Germany.
Gottschalk
U
U
Guntermann
D
D
Hansen
J
J
Mässen
J
J
Schmidt
E
E
Schneider
K H
KH
Schneidereit
M
M
Rübsamen-Waigmann
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D002213:Capsid / Q000235:genetics* / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification; D014764:Viral Proteins
D004274:DNA, Recombinant; D014764:Viral Proteins; C079287:scaffold protein, Herpes simplex virus-1; D012697:Serine Endopeptidases