title: 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.affiliation: Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Viet Nam.
authors.forename: Hong-Loan Thi
authors.initials: HL
authors.lastname: Nguyen
authors.affiliation: Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Viet Nam.
authors.forename: Thuy Thi
authors.initials: TT
authors.lastname: Nguyen
authors.affiliation: Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Viet Nam.
authors.forename: Quy Thi
authors.initials: QT
authors.lastname: Vu
authors.affiliation: Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Viet Nam.
authors.forename: Hang Thi
authors.initials: HT
authors.lastname: Le
authors.affiliation: Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Viet Nam.
authors.forename: Yen
authors.initials: Y
authors.lastname: Pham
authors.affiliation: Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Viet Nam.
authors.forename: Phuong Le
authors.initials: PL
authors.lastname: Trinh
authors.affiliation: Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Viet Nam.
authors.forename: Thuan Phuong
authors.initials: TP
authors.lastname: Bui
authors.affiliation: Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Viet Nam. Electronic address: phantuannghia@vnu.edu.vn.
authors.forename: Tuan-Nghia
authors.initials: TN
authors.lastname: Phan
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015658:HIV Infections / Q000821:virology; D016333:HIV Protease / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1
keywords: HIV-1 protease; Inclusion bodies; Purification; pET32a(+)