[en] HIV-1 is an infectious virus that causes acquired immunodeficiency syndrome (AIDS) and it is one of the major causes of deaths worldwide. The production of HIV-1 protease (PR) on a large scale has been a problem for scientists due to its cytotoxicity, low yield, insolubility, and low activity. HIV-1 C-SA protease has been cloned, expressed, and purified previously, however, with low recovery (0.25 mg/L). Herein we report an optimal expression and solubilisation procedure to recover active HIV-1 C-SA protease enzyme from inclusion bodies. The HIV protease was expressed in seven different vectors (pET11b, pET15b, pET28a pET32a, pET39b, pET41b and pGEX 6P-1). The highest expression was achieved when the vector pET32a (Trx tag) was employed. A total of 19.5 mg of fusion protein was refolded of which 5.5 mg of active protease was obtained after cleavage. The free protease had a high specific activity of 2.81 µmoles/min/mg. Interestingly the Trx-fusion protein also showed activity closer (1.24 µmoles/min/mg) to that of the free protease suggesting that the pET32a vector (Trx tag) expressed in BL21(DE3) pLysS provides a more efficient way to obtain HIV-1 protease.
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
Govender, Deidre; 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
Naicker, Tricia; Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban, 4001, South Africa
Lin, Johnson; School of Life Sciences, University of KwaZulu-Natal, Durban, 4001, 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
Kruger, Hendrik G; Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban, 4001, South Africa. Kruger@ukzn.ac.za
Language :
English
Title :
Optimized Procedure for Recovering HIV-1 Protease (C-SA) from Inclusion Bodies.
Publication date :
February 2019
Journal title :
Protein Journal
ISSN :
1572-3887
Publisher :
Springer Science and Business Media, LLC, Netherlands
This study was funded by the National Research Foundation (NRF) (Grant Number 106803).Acknowledgements This study was funded by the National Research Foundation (NRF) (Grant Number 106803).
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