Optimized Procedure for Recovering HIV-1 Protease (C-SA) from Inclusion Bodies

• Published in: The Protein Journal Vol. 38; no. 1; pp. 30 - 36
• Main Authors: Maseko, Sibusiso B., Govender, Deidre, Govender, Thavendran, Naicker, Tricia, Lin, Johnson, Maguire, Glenn E. M., Kruger, Hendrik G.
• Format: Journal Article
• Language: English
• Published: New York Springer US 15.02.2019; Springer; Springer Nature B.V
• Subjects: Acquired immune deficiency syndrome; AIDS; Animal Anatomy; Biochemistry; Bioorganic Chemistry; Chemistry; Chemistry and Materials Science; Cytotoxicity; Escherichia coli - chemistry; Escherichia coli - genetics; Fusion protein; Histology; HIV; HIV (Viruses); HIV Protease - chemistry; HIV Protease - genetics; HIV Protease - isolation & purification; HIV-1 - enzymology; HIV-1 - genetics; Human immunodeficiency virus; Inclusion bodies; Inclusion Bodies - enzymology; Inclusion Bodies - genetics; Morphology; Organic Chemistry; Protease; Proteases; Proteinase; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Toxicity; Viruses; Fusion tags; Thioredoxin; HIV-1 protease; Inclusion bodies; Refolding
• ISSN: 1572-3887; 1573-4943; 1875-8355
• DOI: 10.1007/s10930-018-9805-7

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.