Expression; HIV-1 (subtype C-SA) protease; Mutants; Purification inclusion bodies; DNA, Recombinant; Recombinant Proteins; HIV Protease; p16 protease, Human immunodeficiency virus 1; Cloning, Molecular/methods; DNA, Recombinant/genetics; Escherichia coli/genetics; HIV Infections/virology; HIV Protease/chemistry; HIV Protease/genetics; HIV Protease/isolation & purification; HIV Protease/metabolism; HIV-1/chemistry; HIV-1/enzymology; HIV-1/genetics; Humans; Inclusion Bodies/genetics; Protein Refolding; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Mutation; Cloning, Molecular; Escherichia coli; HIV Infections; HIV-1; Inclusion Bodies; Biotechnology
Abstract :
[en] 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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Maseko, Sibusiso Bonginkhost ; Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa
Natarajan, Satheesh; Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa
Sharma, Vikas; School of Life Sciences, University of KwaZulu-Natal, Durban 4001, South Africa
Bhattacharyya, Neelakshi; Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa
Govender, Thavendran; Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa
Sayed, Yasien; Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Wits, 2050, South Africa
Maguire, Glenn E M; 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
Lin, Johnson; School of Life Sciences, University of KwaZulu-Natal, Durban 4001, South Africa
Kruger, Hendrik G ; Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa. Electronic address: kruger@ukzn.ac.za
Language :
English
Title :
Purification and characterization of naturally occurring HIV-1 (South African subtype C) protease mutants from inclusion bodies.
We thank the NRF , UKZN , Asphen Pharmacare and WITS for financial support. The protease sequence was supplied by Professor Lynn Morris (Head: HIV Research, National Institute for Communicable Diseases, South Africa).
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