Human immunodeficiency virus, or HIV, is the cause of the acquired immunodeficiency syndrome (AIDS). HIV-1 protease (HIV-1 PR) is a retroviral aspartyl protease (retropepsin) that is crucial for the HIV’s life-cycle. It cleaves freshly synthesized viral polyproteins at the suitable locations into functional protein products as mature protein components of an infectious HIV virion. The ability of the virus to replicate and infect additional cells is disrupted by the mutation of HIV-1 PR’s active site or inhibition of its activity.
HIV-1 PR is believed to be a potential target for designing inhibitors, and therefore, significant focus has been put into its purification and expression in recombinant form. The expression of HIV-1 PR in Escherichia coli has shown to be challenging due to the problems associated with its low expression, yields, and solubility, which have restricted the growth of HIV-1 PR-based activity/inhibitor screening assays. At BioVision, these issues associated with HIV-1 PR are effectively resolved by expressing it as a GST-fusion protein in E. coli at comparatively high yields of the purified fusion protein (5–7 mg/L of cell culture).
The purified protease is highly active and it was effectively employed in the development of a protease-based assay using its proteolytic activity against an HIV-1 PR-specific fluorogenic peptide substrate. In the presence of an aspartyl protease inhibitor (Pepstatin A), the enzyme exhibited a characteristic inhibition of its specific proteolytic activity.
The HIV-1 PR assay is simple, high-throughput adaptable and sensitive. The assay offers a useful tool for screening potential HIV-1 PR inhibitors for chemotherapeutic applications.
HIV-1 Protease: Major Goals of the Project
- Develop an assay for testing the activity of purified HIV-PR and for screening potential HIV-1 PR
- Examine the kinetics of HIV-PR with a particular substrate
- Increase the solubility, expression levels, and yields of HIV-1 PR in E.coli (>1 mg per L of cell culture)
Expression of HIV-1 Protease in E. coli with Different Affinity Tags
Figure 1. HIV-1 PR was expressed in E. coli using different affinity tags: (A) N-terminal Poly-his tag, (B) N-terminal H6 -tag, (C) N-terminal GST- and C-terminal H6 -tag, and (D) C-terminal H6 -tag. Blue colored arrows indicate the corresponding position of the fusion protein on the SDS-PAGE.
Purification and Characterization of HIV-1 Protease
Figure 2. HIV-1 PR was expressed under proprietary growth conditions and purified by suitable chromatographic techniques. After purification, the enzyme was characterized by SDS-PAGE and SEC analyses. (A) SDS-PAGE (4-20%) of purified HIV1-PR; M: Protein Marker, 1: HIV1-PR (10 μg), 2: HIV1-PR (20 μg) and 3: HIV1-PR (30 μg). (B) SEC characterization of purified HIV-PR. It runs at ~31 kDa during SEC and SDS-PAGE analyses.
Kinetics of HIV-1 Protease with a HIV-1 Protease-Specific Substrate and Development of Protease-Based Assay Kits
Figure 3. (A) BioVision’s HIV-1 PR follows a typical Michaelis-Menten Kinetics with the HIV-1 PR-specific proprietary substrate with Km of 9.5 µM. (B) Progressive kinetic curves of different amounts of HIV-1 PR are linear for an extended period of time (1-3 h), indicating the stability of the enzyme under assay conditions. (C) HIV-PR yields a measurable fluorescence even within 1 h of assay. (D) HIV-PR activity was abolished by the presence of Pepstatin A inhibitor.
Market's First and Only HIV-1 Protease Inhibitor Screening Kit (Fluorometric)
Figure 4. Inhibition of HIV1- PR activity by HIV-1 PR Inhibitor (Pepstatin A). Inhibition curves were plotted in Graphpad Prism 5 and IC50 was found to be 1.6 µM which is consistent with earlier literature value (0.7 µM)*.
(*Kräusslich et. al.,” Activity of purified biosynthetic proteinase of human immunodeficiency virus on natural substrates and synthetic peptides”, Proc. Natl. Acad. Sci. U S A. 1989 Feb; 86(3): 807–811)
- HIV-1 PR was effectively expressed and purified as a GST-fusion protein in E. coli at comparatively high yields of the purified fusion protein (5–7 mg/L of cell culture)
- Characterization of HIV-PR with a HIV-1 PR-specific substrate signified a Michaelis-Menten Kinetics
- HIV-1 PR enzyme was effectively employed to develop inhibitor screening kits and protease-based activity assay
- HIV-1 PR assays are simple, high-throughput adaptable, and sensitive
Key Features of HIV-1 Protease Kits
- Fluorometric Detection and Quantitation (Non-Radioactive), (Ex/Em = 330/450 nm)
- Fast and simple, and High-Throughput adaptable
- Specific: HIV-1 PR specific substrate
- Sufficient reagents to execute 100 assays in a 96-well plate format
- Extremely Stable: Long shelf life
BioVision, Inc., is a privately held Life Science company headquartered in the beautiful San Francisco Bay Area.
BioVision develops and offers a wide variety of products including assay kits, antibodies, recombinant proteins & enzymes, and other innovative research tools for studying Apoptosis, Metabolism, Cell Proliferation, Cellular Stress, Cell Damage and Repair, Diabetes, Obesity and Metabolic Syndrome, Stem Cell Biology, Gene Regulation, Signal Transduction, etc. BioVision's products are currently being sold in more than 60 countries worldwide.
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