An efficient procedure for the expression and purification of HIV-1 protease from inclusion bodies.

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.

• Authors: Hong-Loan Thi Nguyen, Thuy Thi Nguyen, Quy Thi Vu, Hang Thi Le, Yen Pham, Phuong Le Trinh, Thuan Phuong Bui, Tuan-Nghia Phan
• Journal: Protein expression and purification (Protein Expr Purif) Vol. 116 Pg. 59-65 (Dec 2015) ISSN: 1096-0279 [Electronic] United States
• PMID: 26231073 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2015 Elsevier Inc. All rights reserved.

Chemical References

• Recombinant Fusion Proteins
• HIV Protease
• p16 protease, Human immunodeficiency virus 1

Topics

• Amino Acid Sequence
• Base Sequence
• Chromatography, Affinity
• Chromatography, Ion Exchange
• Cloning, Molecular (methods)
• Electrophoresis, Polyacrylamide Gel
• Escherichia coli (genetics)
• HIV Infections (virology)
• HIV Protease (chemistry, genetics, isolation & purification, metabolism)
• HIV-1 (chemistry, enzymology, genetics)
• Humans
• Inclusion Bodies (genetics)
• Molecular Sequence Data
• Plasmids (genetics)
• Protein Refolding
• Recombinant Fusion Proteins (chemistry, genetics, isolation & purification, metabolism)