In recent years, various strategies have been used to overexpress and purify HIV-1 protease because it is an essential drug
target in anti-retroviral therapy. Obtaining sufficient quantities of the enzyme, however, remains challenging. Overexpression
of large quantities is prevented due to the enzyme’s autolytic nature and its inherent cytotoxicity in Escherichia coli
cells. Here, we describe a novel HIV-1 protease purification method using a thioredoxin–hexahistidine fusion system for
the wild-type and two variant proteases. The fusion proteases were overexpressed in E. coli and recovered by immobilised
metal ion affinity chromatography. The proteases were cleaved from the fusion constructs using thro... More
In recent years, various strategies have been used to overexpress and purify HIV-1 protease because it is an essential drug
target in anti-retroviral therapy. Obtaining sufficient quantities of the enzyme, however, remains challenging. Overexpression
of large quantities is prevented due to the enzyme’s autolytic nature and its inherent cytotoxicity in Escherichia coli
cells. Here, we describe a novel HIV-1 protease purification method using a thioredoxin–hexahistidine fusion system for
the wild-type and two variant proteases. The fusion proteases were overexpressed in E. coli and recovered by immobilised
metal ion affinity chromatography. The proteases were cleaved from the fusion constructs using thrombin. When compared
to the standard overexpression and purification protocol in use in our laboratory, the expression of the fusion-derived wildtype
protease was increased from 0.83 to 2.5 mg/l of culture medium. The expression levels of the two variant proteases
ranged from 1.5 to 2 mg/l of culture medium. The fusion wild-type and variant proteases were inactive before the cleavage
of the thioredoxin–hexahistidine fusion tag as no enzymatic activity was observed. The proteases were, however, active after
cleavage of the tag. The novel thioredoxin–hexahistidine fusion system, therefore, enables the successful overexpression and
purification of catalytically active HIV-1 proteases.