Avidin-biotin catch principle has been widely used for affinity purification methods of proteins. In order to analyze activity of the purified proteases, some procedures such as removing denaturant from elution buffer and refolding are needed. Our research group previously reported synthesis of inhibitors with longer linker and terminal biotin targeting HIV protease and malarial plasmepsin, high recovery in affinity purification from a protein mixture. Streptavidin-linked column was used for increasing the amount of the bound protein, but the centrifugation steps for concentration and buffer exchange damaged the conformation of the eluted protein. To test the protease activity of the eluted protein directly, magnetic beads carrier would be preferable using small volume for elution without centrifugation.
We synthesize novel biotinylated inhibitors. Extension of linker and coupling of biotin to amino group of allophenylnorstatine-containing inhibitors was performed by conventional peptide synthesis strategy. These biotinylated inhibitors were highly potent against HIV-1 protease at nanomolar level. These compounds were bound to commercially available streptavidin magnetic beads. After affinity purification, the eluate was directly added to enzyme assay buffer. We eluted recombinant aspartic proteases in serum-containing culture medium successfully to detect FRET substrate fragment. This methodology was applied to biotinylated pepstatin A. These results suggest that activity detection using the presented method is relatively easy and would be new tool to directly analyze function of affinity-purified proteases.