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An efficient procedure for the expression and purification of HIV-1 protease from inclusion bodies.
- Source :
-
Protein Expression & Purification . Dec2015, Vol. 116, p59-65. 7p. - Publication Year :
- 2015
-
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]
Details
- Language :
- English
- ISSN :
- 10465928
- Volume :
- 116
- Database :
- Academic Search Index
- Journal :
- Protein Expression & Purification
- Publication Type :
- Academic Journal
- Accession number :
- 110510620
- Full Text :
- https://doi.org/10.1016/j.pep.2015.07.011