Journal
PROTEIN JOURNAL
Volume 37, Issue 4, Pages 369-379Publisher
SPRINGER
DOI: 10.1007/s10930-018-9779-5
Keywords
HIV-1; Protease; Escherichia coli; Metal ion affinity chromatography; Fusion protein; Hexahistidine tag
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Funding
- University of the Witwatersrand, South African National Research Foundation [68898]
- South African Research Chairs Initiative of the Department of Science and Technology
- National Research Foundation [64788]
- South African Medical Research Council (SAMRC) under a Self-Initiated Research Grant
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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 wild-type 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.
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