Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban, 4001, South Africa.
School of Life Sciences, University of KwaZulu-Natal, Durban, 4001, South Africa.
Protein J. 2019 Feb;38(1):30-36. 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.
HIV-1 是一种传染性病毒，可导致获得性免疫缺陷综合征（AIDS），是全球主要死亡原因之一。由于 HIV-1 蛋白酶（PR）具有细胞毒性、产量低、不溶性和低活性，因此大规模生产一直是科学家面临的问题。此前，HIV-1 C-SA 蛋白酶已被克隆、表达和纯化，但回收率低（0.25mg/L）。在此，我们报告了一种从包涵体中回收有活性的 HIV-1 C-SA 蛋白酶的最佳表达和溶解程序。HIV 蛋白酶在七种不同载体（pET11b、pET15b、pET28a、pET32a、pET39b、pET41b 和 pGEX6P-1）中进行表达。当使用载体 pET32a（Trx 标签）时，表达量最高。共折叠了 19.5mg 的融合蛋白，经切割后获得了 5.5mg 的活性蛋白酶。游离蛋白酶具有 2.81 µmoles/min/mg 的高比活。有趣的是，Trx 融合蛋白的活性也接近游离蛋白酶（1.24 µmoles/min/mg），这表明 BL21(DE3)pLysS 中表达的 pET32a 载体（Trx 标签）提供了一种更有效的获得 HIV-1 蛋白酶的方法。
