A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening

Abstract

In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection.

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

“…Furthermore, the substrates contain new generation fluorescent proteins, which are highly stable and have a monomeric form to prevent substrate aggregation. Besides the previously published application of mTurquoise2-and mApple-fused forms 14 , here we also show results given by the use of a recombinant substrate containing a monomeric enhanced yellow fluorescent protein (mEYFP) fluorescent tag. Hereby we demonstrate the compatibility of the system with other fluorescent proteins and represent some general types of results that can be acquired by the protease assay.…”

“…NOTE: It is recommended to use a cleavage buffer to dissolve and/or dilute the enzyme. Protocols for the purification of HIV-1 14 and TEV PRs 18 have been published previously.…”

“…In contrast to the previously described methods, in the system presented here, the amounts of substrate and C-terminal cleavage products are uniquely quantified based on a detailed substrate calibration procedure. The assay system can be supported by an SDS-PAGE analysis of the assay samples; a subsequent fluorescent in-gel visualization may be applied immediately after the electrophoresis or after the in-gel renaturation of the nondenatured and denatured fluorescent components, respectively 14 .…”

Biochemical characterization of Ty1 retrotransposon protease

“…We used a slightly modified pDest-His 6 -MBP-mTurquoise2 plasmid, prepared in our laboratory by Gateway Cloning Technology as previously described [31], and modified in the present study as follows. The empty pDest-His 6 -MBP-mTurquoise2 plasmid was linearized by PacI and NheI endonucleases (New England Biolabs).…”

“…Recombinant fluorescent substrates were expressed in E. coli BL21(DE3) cells as previously reported [31][32][33]. The His 6 -tagged fluorescent recombinant protein substrates were purified from the supernatant of the lysed cells by the addition of Ni-NTA magnetic agarose beads (Qiagen) and were incubated for 30 min while continuously shaking.…”