(474b) Development of a Genetically Programmable Protein Module for High Throughput Screening of HIV-1 Protease Inhibitors
AIChE Annual Meeting
2009
2009 Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Protein Engineering III - Applications
Wednesday, November 11, 2009 - 3:35pm to 3:55pm
Human immunodeficiency virus type 1 protease (HIV-1 PR) plays a key role in viral maturation and production of infectious virus particles by proteolytic cleavage of viral polyproteins, namely Gag and Gag-Pol polyproteins, and is thus a major target for anti-HIV agents. However, one of the emerging problem with antiviral therapy is the emergence of drug resistant variants. Thus, it is imperative at this point to identify small molecule inhibitors that could block PR cleavage by selectively inhibiting cleavage at a particular site. Here, we demonstrate the development of a protein expression module to rapidly express, purify and introduce the probes inside living cells with minimal cytotoxic effects, for probing HIV-protease activity and screening its inhibitors. A cell based platform allows monitoring the transport efficiency and cytotoxicity of the inhibitors in the cells. The genetically engineered protein module is designed with: 1) a QD binding moiety containing poly-histidine, 2) a cysteine site for fluorescent dye incorporation based on cysteine-thiol interaction, 3) a protease cleavage site, and 4) an ELP domain for thermal purification and 5) a flanking TAT peptide sequence for cell penetration. In this design, the QD photoluminescence is quenched by the fluorescent dye due to fluorescence resonance energy transfer (FRET). Cleavage of the sequence by HIV-1 PR resulted in separation of the FRET pair with subsequent increase in QD fluorescence, whereas potent protease inhibitors prevented FRET disruption and a reduced QD fluorescence was detected. HIV-1 PR was expressed by transiently transfecting cells with a non-replicative HIV-1 molecular vector. Thus, the genetically programmable protein module as QD-based FRET substrates can be used as a convenient, cost effective and highly efficient system to do high throughput screening of viral protease inhibitors.