Detection of Proteolysis Using FCCS

Introduction

When mKeima-Red is fused to mAG1 with a linker containing protease recognition peptide sequence, the protease activation will be detected through the decrease in the cross-correlation amplitude. Here, the C terminus of mKeima-Red and the N terminus of mAG1 were linked using a peptide containing the trypsin cleavage sequence. The recombinant protein (mKeima-Red-linker-mAG1) was examined to confirm whether trypsin activity could be detected.

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Substance


The recombinant protein (His-tag-mKeima-Red-linker-mAG1) from E. coli was purified using Ni-NTA agarose.

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Measurement

A recombinant protein (~20nM, buffer: 150 mM KCl, 50 mM HEPES KOH pH 7.4) was mixed with trypsin. Measurements were made every few minutes.

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Result

The activity of trypsin was detected using FCCS. The degree of cross-correlation of mKeima-Red and mAG1 decreased due to the addition of trypsin.


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