title: Purification and characterization of naturally occurring HIV-1 (South African subtype C) protease mutants from inclusion bodies.
abstract: Human immunodeficiency virus (HIV) infections in sub-Saharan Africa represent about 56% of global infections. Many studies have targeted HIV-1 protease for the development of drugs against AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. Along with the wild type (C-SA) we also over-expressed and characterized two mutant forms from patients that had shown resistance to protease inhibitors. Using recombinant DNA technology, we constructed three recombinant plasmids in pGEX-6P-1 and expressed them containing a sequence encoding wild type HIV protease and two mutants (I36T↑T contains 100 amino acids and L38L↑N↑L contains 101 amino acids). These recombinant proteins were isolated from inclusion bodies by using QFF anion exchange and GST trap columns. In SDS-PAGE, we obtained these HIV proteases as single bands of approximately 11.5, 11.6 and 11.7 kDa for the wild type, I36T↑Tand L38L↑N↑L mutants, respectively. The enzyme was recovered efficiently (0.25 mg protein/L of Escherichia coli culture) and had high specific activity of 2.02, 2.20 and 1.33 μmol min(-1) mg(-1) at an optimal pH of 5 and temperature of 37 °C for the wild type, I36T↑T and L38L↑N↑L, respectively. The method employed here provides an easy and rapid purification of the HIV-1(C-SA) protease from the inclusion bodies, with high yield and high specific activities.
authors.affiliation: Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa.
authors.forename: Sibusiso B
authors.initials: SB
authors.lastname: Maseko
authors.affiliation: Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa.
authors.forename: Satheesh
authors.initials: S
authors.lastname: Natarajan
authors.affiliation: School of Life Sciences, University of KwaZulu-Natal, Durban 4001, South Africa.
authors.forename: Vikas
authors.initials: V
authors.lastname: Sharma
authors.affiliation: Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa.
authors.forename: Neelakshi
authors.initials: N
authors.lastname: Bhattacharyya
authors.affiliation: Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa.
authors.forename: Thavendran
authors.initials: T
authors.lastname: Govender
authors.affiliation: Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Wits, 2050, South Africa.
authors.forename: Yasien
authors.initials: Y
authors.lastname: Sayed
authors.affiliation: Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa; School of Chemistry and Physics, University of KwaZulu-Natal, Durban 4001, South Africa.
authors.forename: Glenn E M
authors.initials: GE
authors.lastname: Maguire
authors.affiliation: School of Life Sciences, University of KwaZulu-Natal, Durban 4001, South Africa.
authors.forename: Johnson
authors.initials: J
authors.lastname: Lin
authors.affiliation: Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa. Electronic address: kruger@ukzn.ac.za.
authors.forename: Hendrik G
authors.initials: HG
authors.lastname: Kruger
mesh_terms: D003001:Cloning, Molecular / Q000379:methods; D004274:DNA, Recombinant / Q000235:genetics; 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; D009154:Mutation; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D004274:DNA, Recombinant; D011994:Recombinant Proteins; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1
keywords: Expression; HIV-1 (subtype C-SA) protease; Mutants; Purification inclusion bodies