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 simple 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 a utoproteolytic processing between residues Ala247/Ser248 and Ala610/Se r611. The N-terminal processing product (amino acids 1-247) contains t he protease domain. To perform crystallization studies and high throug hput screening for potent inhibitors, large amounts of the HSV-1 prote ase are required. However, expression of the natural HSV-1 protease ge ne in Escherichia coli using a T7-promoter-regulated system is low and does not allow for the efficient production of larger amounts of high ly purified enzyme. In this report, we describe the use of a synthetic protease gene with optimized E. coli codon usage. The level of protea se 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 hydroxapatite chromatography.