Development of HIV-1 protease expression methods using the T7 phage promoter system

Area of Science:

• Biotechnology
• Molecular Biology
• Virology

Background:

• Human immunodeficiency virus type 1 (HIV-1) protease is a crucial target for antiviral therapies.
• Efficient expression of recombinant proteins, especially those toxic to host cells like HIV-1 protease, remains a challenge in biotechnology.
• The T7 phage promoter system is widely used for high-level protein expression in Escherichia coli (E. coli).

Purpose of the Study:

• To develop novel and simplified methods for expressing human immunodeficiency virus type 1 (HIV-1) protease in E. coli.
• To suppress leaky expression of HIV-1 protease controlled by the T7 polymerase.
• To enhance the overall activity of expressed HIV-1 protease.

Main Methods:

• Development of two new methods to suppress leaky expression of HIV-1 protease in E. coli using the T7 promoter system.
• Method 1: Introduction of supplementary T7 promoter regions into E. coli BL-21 (DE3) to compete for T7 polymerase binding.
• Method 2: Infection of E. coli JM109 with M13 phage expressing T7 polymerase in late-log-phase cultures.

Main Results:

• Both developed methods effectively suppressed leaky expression and enhanced HIV-1 protease activity.
• Transformants utilizing the new expression systems exhibited a tenfold increase in HIV-1 protease activity compared to the autoprocessing vector.
• The new expression systems proved convenient and adaptable for other proteins toxic to E. coli.

Conclusions:

• The developed T7 promoter-based expression systems offer a significant improvement for producing active HIV-1 protease in E. coli.
• These methods provide a robust and efficient platform for expressing proteins that are otherwise toxic to the host E. coli.
• The simplicity and applicability of these systems make them valuable tools in biotechnology and molecular biology research.