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HIV-I protease. Cloning, expression, and purification
N I Dergousova1, Amerik AYu, A M Volynskaya
1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia. nd@enzyme.siobc.ras.ru
Applied Biochemistry and Biotechnology
|October 1, 1996
Summary
This study presents a novel method for producing HIV-I protease using fusion proteins in E. coli. Optimized cleavage and purification techniques yielded active HIV-I protease for further research.
Area of Science:
- Biochemistry
- Molecular Biology
- Virology
Background:
- Human Immunodeficiency Virus type 1 (HIV-I) protease is a critical enzyme for viral maturation.
- Efficient production of active HIV-I protease is essential for drug development and structural studies.
- Previous methods for obtaining HIV-I protease have limitations in yield and purity.
Purpose of the Study:
- To develop a novel and efficient method for the recombinant expression and purification of active HIV-I protease.
- To investigate different linker strategies and cleavage conditions for obtaining soluble and active protease.
- To characterize the enzymatic activity of the purified HIV-I protease.
Main Methods:
- Expression of fusion proteins (N-terminal fragment of human gamma-interferon with HIV-I protease) in E. coli.
- Utilized (Asp)4Lys (Protein I) and Asp-Pro (Protein II) linkers.
- Investigated enterokinase cleavage for Protein I and acidic hydrolysis for Protein II.
- Optimized denaturing conditions with Na-sulfite/Na-tetrathionate for Protein I solubility.
- Separated hydrolysis products using reversed-phase FPLC.
- Assessed enzyme activity using chromogenic and high-molecular-weight substrates.
Main Results:
- Fusion proteins were expressed as insoluble inclusion bodies, yielding 20% of total cell protein.
- Protein I solubility was enhanced using Na-sulfite/Na-tetrathionate treatment.
- Optimal acidic hydrolysis conditions for the Asp-Pro bond in Protein II were determined.
- Purified HIV-I protease was obtained and its activity was confirmed using various substrates.
- Amino acid composition (tryptophan, cysteine) of the obtained enzyme was estimated.
Conclusions:
- A new method for obtaining active HIV-I protease has been successfully developed.
- The described fusion protein expression and cleavage strategies offer a viable approach for protease production.
- The characterized HIV-I protease is suitable for further biochemical and structural investigations.