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Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation
JoVE Journal
Biochemistry
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JoVE Journal Biochemistry
Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

DOI:
10.3791/58824-v

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19:23 min

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January 16, 2019

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, , , ,
1Department of Biochemistry and Molecular Biology, Faculty of Medicine,University of Debrecen, 2Biotechnology Research Department,Gedeon Richter Plc

Chapters

  • 00:00Title
  • 01:05Generation of the Substrate-coding Expression Plasmids
  • 02:16Expression of the Fluorescent Substrates
  • 03:35Cell Disruption
  • 04:26Ni-NTA Magnetic Agarose Bead-based Protease Assay
  • 12:39PAGE Analysis
  • 14:41Representative Results
  • 18:29Conclusion

Summary

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Here, we present the detailed procedure of a recently developed protease assay platform utilizing N-terminal hexahistidine/maltose-binding protein and fluorescent protein-fused recombinant substrates attached to the surface of nickel-nitrilotriacetic acid magnetic agarose beads. A subsequent in-gel analysis of the assay samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is also presented.

Tags

Keywords: Recombinant Fusion ProteinsFluorescent Protease AssayIn-gel RenaturationHIV-1 ProteaseHigh-throughput ScreeningFluorescence-based AssayProtein ExpressionCleavage SiteBL21(DE3) CellsPlasmid Construction

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