1. An efficient procedure for the expression and purification of HIV-1 protease from inclusion bodies.
- Author
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Nguyen, Hong-Loan Thi, Nguyen, Thuy Thi, Vu, Quy Thi, Le, Hang Thi, Pham, Yen, Trinh, Phuong Le, Bui, Thuan Phuong, and Phan, Tuan-Nghia
- Subjects
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PROTEIN expression , *CHEMICAL purification , *PROTEOLYTIC enzymes , *CELLULAR inclusions , *HIV infections , *THERAPEUTICS , *DRUG development - Abstract
Several studies have focused on HIV-1 protease for developing drugs for treating AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. However, large-scale expression and purification of this enzyme is difficult mainly because of its low expression and solubility. In this study, we constructed 9 recombinant plasmids containing a sequence encoding HIV-1 protease along with different fusion tags and examined the expression of the enzyme from these plasmids. Of the 9 plasmids, pET32a(+) plasmid containing the HIV-1 protease-encoding sequence along with sequences encoding an autocleavage site GTVSFNF at the N-terminus and TEV plus 6× His tag at the C-terminus showed the highest expression of the enzyme and was selected for further analysis. The recombinant protein was isolated from inclusion bodies by using 2 tandem Q- and Ni-Sepharose columns. SDS–PAGE of the obtained HIV-1 protease produced a single band of approximately 13 kDa. The enzyme was recovered efficiently (4 mg protein/L of cell culture) and had high specific activity of 1190 nmol min −1 mg −1 at an optimal pH of 4.7 and optimal temperature of 37 °C. This procedure for expressing and purifying HIV-1 protease is now being scaled up to produce the enzyme on a large scale for its application. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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