Two‐Step Preparation of Highly Pure, Soluble HIV Protease from Inclusion Bodies Recombinantly Expressed in Escherichia coli. (27th April 2020)

• Record Type: Journal Article
• Authors: Sherry, Dean; Worth, Roland; Sayed, Yasien
• Editors: Coligan, John E.; Dunn, Ben M.; Speicher, David W.; Wingfield, Paul T.
• Is Part Of: Current protocols in protein science. Volume 100(2020)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2020-04-27
• Subjects: HIV protease -- inclusion bodies -- ion‐exchange chromatography -- protein purification -- protein refolding; Proteins -- Laboratory manuals; Proteins; Clinical Laboratory Techniques; Genetic Techniques; Immunologic Techniques; Proteins; Laboratory manuals; 572.6028
• DOI: 10.1002/cpps.106
• Languages: English
• ISSNs: 1934-3655

Abstract

Abstract: Heterologous expression of exogenous proteases in Escherichia coli often results in the formation of insoluble inclusion bodies. When sequestered into inclusion bodies, the functionality of the proteases is minimized. To be characterized structurally and functionally, however, proteases must be obtained in their native conformation. HIV protease is readily expressed as inclusion bodies, but must be recovered from the inclusion bodies. This protocol describes an efficient method for recovering HIV protease from inclusion bodies, as well as refolding and purifying the protein. HIV protease–containing inclusion bodies are treated with 8 M urea and purified via cation‐exchange chromatography. Subsequent refolding by buffer exchange via dialysis and further purification by anion‐exchange chromatography produces highly pure HIV protease that is functionally active. © 2020 by John Wiley & Sons, Inc. Basic Protocol : Recovery, refolding, and purification of HIV protease from inclusion bodies Support Protocol 1 : Expression and extraction of inclusion bodies containing HIV protease expressed in Escherichia coli Support Protocol 2 : Determination of the active site concentration of HIV protease via isothermal titration calorimetry