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Expression and purification of Active Recombinant HIV-1 Protease and the development of protease-based activity/inhibitor screening assays
EP27366
Poster Title: Expression and purification of Active Recombinant HIV-1 Protease and the development of protease-based activity/inhibitor screening assays
Submitted on 15 Mar 2018
Author(s): Santoshkumar L. Khatwani, Ph.D., Grigoriy Tchaga, Ph.D. and Gordon Yan, Ph.D.
Affiliations: BioVision Inc.
Poster Views: 612
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Poster Information
Abstract: Human Immunodeficiency Virus (HIV) is the cause of the Acquired Immunodeficiency
Syndrome (AIDS). HIV-1 protease (HIV-1 PR) is a retroviral aspartyl protease
(retropepsin) that is essential for the life-cycle of HIV. It cleaves newly synthesized viral
polyproteins at the appropriate places into functional protein products as mature
protein components of an infectious HIV virion. The mutation of HIV-1 PR's active site
or inhibition of its activity disrupts the ability of the virus to replicate and infect
additional cells. HIV-1 PR is considered an attractive target for designing inhibitors and
considerable focus has been put into its expression and purification in recombinant
form. The expression of HIV-1 PR in Escherichia coli has proven to be difficult due to
the issues related to its low expression, solubility and yields, which have limited the
development of HIV-1 PR-based activity/inhibitor screening assays. At BioVision, we
have successfully overcome these issues associated with HIV-1 PR by expressing it
as a GST-fusion protein in E. coli at relatively high yields of the purified fusion protein
(5-7 mg/L of cell culture). The purified protease is highly active and it was
successfully utilized in developing a protease-based assay using its proteolytic activity
against an HIV-1 PR-specific fluorogenic peptide substrate. The enzyme showed
characteristic inhibition of its specific proteolytic activity in the presence of an aspartyl
protease inhibitor (Pepstatin A). The HIV-1 PR assay is sensitive, simple, and highthroughput
adaptable. The assay provides a useful tool for screening of potential HIV-1
PR inhibitors for chemotherapeutic applications.
Summary: 1. HIV-1 PR was successfully expressed and purified as a GST-fusion protein in E. coli
at relatively high yields of the purified fusion protein (5-7 mg/L of cell culture).
2. Characterization of HIV-PR with a HIV-1 PR-specific substrate indicated a MichaelisMenten
Kinetics.
3. HIV-1 PR enzyme was successfully utilized to develop protease-based activity
assay and inhibitor screening kits.
4. HIV-1 PR assays are sensitive, simple, high-throughput adaptable
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