Improved Production of HIV-1 Subtype C Protease from Transgenic E. Coli

Abstract

Background:Human Immunodeficiency Virus 1 (HIV-1) subtype C is responsible for the majority of infections of patients in Southern Africa. The HIV protease is a primary target for the development of highly efficient anti-retroviral pharmaceuticals because of its pivotal role in the maturation of the virus in the host cell. For target validation of novel HIV protease inhibitors, there is a need for the availability of an abundance of this protease.Objective:This study reports an optimized method to produce HIV-1 protease derived from HIV-1 subtype C.Methods:It involves the use of a transgenicE. colistrain that overexpresses the native form of the enzymeviainclusion bodies. A stringent method for the isolation, purification, and renaturation resulted in the production of highly pure active HIV-1 protease. In order to facilitate an increase in protease yields, an optimized growth strategy was developed. In this regard, a chemically defined medium with lower glucose content and devoid of essential amino acids of the TCA cycle was used as an alternative to the widely used nutrient-rich Luria Bertani (LB) medium.Results:Results indicated an increase in protease yield up to twice the amount, thereby making this medium an attractive alternative for increasing biomass and HIV protease production for future research.Conclusion:An optimized method for HIV-1 protease derived from HIV-1 subtype C production using chemically defined media was established. This was achieved using a known method to isolate and purify the enzyme with the use of a specialized feeding strategy.