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

Protein J. 2019 Feb;38(1):30-36. doi: 10.1007/s10930-018-9805-7.

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.

Keywords: Fusion tags; HIV-1 protease; Inclusion bodies; Refolding; Thioredoxin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Escherichia coli / chemistry
  • Escherichia coli / genetics
  • HIV Protease / chemistry*
  • HIV Protease / genetics
  • HIV Protease / isolation & purification*
  • HIV-1 / enzymology*
  • HIV-1 / genetics
  • Inclusion Bodies / enzymology*
  • Inclusion Bodies / genetics
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification

Substances

  • Recombinant Proteins
  • HIV Protease
  • p16 protease, Human immunodeficiency virus 1