H.L.T., Nguyen, Hong Loan ThiNguyen, Hong Loan ThiH.L.T.T.T., Nguyen, Thuy ThiNguyen, Thuy ThiT.T.Q., Vu, QuythiVu, QuythiQ.H.T., Le, Hang ThiLe, Hang ThiH.T.Y.B., Pham, Yen BaoPham, Yen BaoY.B.P.L., Trinh, Phuong LeTrinh, Phuong LeP.L.Bui Phuong, ThuanThuanBui PhuongPhan, T. N.T. N.Phan2025-09-142025-09-14201510.1016/j.pep.2015.07.011https://scholar.vnu.edu.vn/handle/123456789/8662Several 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. © 2015 Elsevier B.V., All rights reserved.EnglishHiv-1 ProteaseInclusion BodiesPet32a(+)PurificationHuman Immunodeficiency Virus ProteinaseHiv ProteaseP16 Protease, Human Immunodeficiency Virus 1Recombinant Fusion ProteinsHuman Immunodeficiency Virus ProteinaseHybrid ProteinP16 Protease, Human Immunodeficiency Virus 1Affinity ChromatographyAmino Acid SequenceCell InclusionChemistryEnzymologyEscherichia ColiGeneticsHumanHuman Immunodeficiency Virus 1Human Immunodeficiency Virus InfectionIon Exchange ChromatographyIsolation And PurificationMetabolismMolecular CloningMolecular GeneticsNucleotide SequencePlasmidPolyacrylamide Gel ElectrophoresisProceduresProtein RefoldingVirologyAmino Acid SequenceBase SequenceChromatography, AffinityChromatography, Ion ExchangeCloning, MolecularElectrophoresis, Polyacrylamide GelHiv InfectionsHiv ProteaseHiv-1HumansInclusion BodiesMolecular Sequence DataPlasmidsProtein RefoldingRecombinant Fusion ProteinsArticle