Home › Biochemistry › Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation
Biochemistry JoVE (Open Access) Citable · DOI

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

DOI: 10.3791/58824-v
What you'll learn
  • ✓ Design and construct expression plasmids encoding N-terminal His-MBP-fused fluorescent protease substrates
  • ✓ Express and purify recombinant fluorescent fusion proteins from bacterial cultures
  • ✓ Perform magnetic bead-based protease activity assays using immobilized substrates
  • ✓ Analyze protease cleavage products via SDS-PAGE and in-gel fluorescence detection
Protocol

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.

Difficulty
advanced
Total time
~3–4 days (plasmid generation ~1 day, protein expression/purification ~1–2 days, assay and analysis ~4–6 hrs)
Biosafety
BSL-1

Steps

1
Generate substrate-coding expression plasmids

Design and construct plasmids encoding N-terminal hexahistidine/maltose-binding protein fused to fluorescent protein substrates. Includes cloning strategy and verification of insert sequences.

▶ 01:05
2
Express fluorescent substrate proteins in bacteria

Culture transformed bacterial cells and induce recombinant protein expression under optimal conditions to generate His-MBP-fluorescent protein fusion substrates.

▶ 02:16
3
Disrupt cells and prepare protein lysate

Harvest induced bacterial cells and lyse them using mechanical or chemical disruption to release soluble recombinant proteins.

▶ 03:35
4
Perform magnetic bead-based protease assay

Immobilize His-tagged substrates on nickel-nitrilotriacetic acid magnetic agarose beads and incubate with protease samples to measure enzymatic activity via fluorescence readout.

▶ 04:26
5
Analyze cleavage products by SDS-PAGE

Separate assay samples on sodium dodecyl sulfate-polyacrylamide gels and perform in-gel fluorescence imaging to visualize intact and cleaved substrate bands.

▶ 12:39
6
Interpret representative results and data

Review example protease assay outputs, including fluorescence patterns, cleavage profiles, and quantitative analysis of protease activity from representative experiments.

▶ 14:41
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