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Optimized Procedure for Recovering HIV-1 Protease (C-SA) from Inclusion Bodies.
Maseko, Sibusiso B; Govender, Deidre; Govender, Thavendran; Naicker, Tricia; Lin, Johnson; Maguire, Glenn E M; Kruger, Hendrik G.
Afiliação
  • Maseko SB; Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban, 4001, South Africa.
  • Govender D; Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban, 4001, South Africa.
  • Govender T; Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban, 4001, South Africa.
  • Naicker T; Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban, 4001, South Africa.
  • Lin J; School of Life Sciences, University of KwaZulu-Natal, Durban, 4001, South Africa.
  • Maguire GEM; Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban, 4001, South Africa.
  • Kruger HG; School of Chemistry and Physics, University of KwaZulu-Natal, Durban, 4001, South Africa.
Protein J ; 38(1): 30-36, 2019 02.
Article em En | MEDLINE | ID: mdl-30666487
ABSTRACT
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.
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Texto completo: 1 Base de dados: MEDLINE Assunto principal: Corpos de Inclusão / Protease de HIV / HIV-1 Idioma: En Ano de publicação: 2019 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Corpos de Inclusão / Protease de HIV / HIV-1 Idioma: En Ano de publicação: 2019 Tipo de documento: Article