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

Abstract

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes. In this assay system, the applied substrates contain N-terminal hexahistidine (His 6 ) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

Biochemical characterization of Ty1 retrotransposon protease

“…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.…”

“…Finally, the magnetic beads binding the His 6 -tagged fluorescent recombinant protein substrates were washed with the following Ty1 cleavage buffers: cleavage buffer A (10 mM PIPES, 75 mM NaCl, 0.25% Nonidet P-40, 5% glycerol, 75 mM KCl, 12.5 mM NaH 2 PO 4 , 61.25 mM sodium-glutamate, 12.5 mM MgSO 4 , 0.125 mM CaCl 2 , 0.05% Tween20, pH 7.0) or cleavage buffer B (50 mM MES, 100 mM Tris, 50 mM sodium-acetate, 150 mM NaCl, 75 mM KCl, 12.5 mM NaH 2 PO 4 , 61.25 mM sodium-glutamate, 12.5 mM MgSO 4 , 0.125 mM CaCl 2 , 0.05% Tween20, pH 8.0). The purified His 6 -tagged fluorescent recombinant protein substrates were used for proteolytic assays, based on the method described previously [31][32][33].…”

“…Uncleaved substrates and cleavage products were separated by SDS-PAGE using 16% SDS gels. The denatured fluorescent proteins were renatured by rinsing the polyacrylamide gel in distilled water to remove SDS, as described previously [31,33]. After in-gel renaturation, both blue light transillumination (Dark Reader transilluminator, Clare Chemical Research) and Coomassie staining (Page-Blue Protein Staining solution, Thermo Scientific) were used for protein detection.…”

Functional Study of the Retrotransposon-Derived Human PEG10 Protease

“…For preparation of a recombinant fusion protein substrate containing the 1-345 residues of the full-length fs RF1/RF2 PEG10 protein (His 6 -MBPfs RF1/RF2 PEG10 (1-345)-mTurquoise2), we used a slightly modified version of the previously described method [51]. The introduced changes are detailed below.…”

“…A pGEX-4T-3 expression plasmid containing the codon-optimized coding sequence of the fs RF1/RF2 PEG10 protein was obtained from GenScript, and the pDest-His 6 -MBP-mTurquoise2 bacterial expression plasmid was used from the in-house stock [51] The coding sequence of the 1-345 region of the fs RF1/RF2 PEG10 protein was amplified using PCR, and both the amplicon and pDest-His 6 -MBP-mTurquoise2 plasmid were digested with PacI and NheI restriction endonucleases followed by ligation. The plasmid was transformed into BL21(DE3) Escherichia coli cells, and then the His 6 -MBPfs RF1/RF2 PEG10 (1-345)-mTurquoise2 protein substrate was expressed at 16 • C for 6 h. After cell lysis, the protein substrate was purified by Ni-NTA magnetic agarose beads (Qiagen, Hilden, Germany), after which the buffer was changed to modified storage buffer (20 mM PIPES, 100 mM NaCl, 0.05% Tween20, pH 7.0) using 0.5 mL 10K Amicon Ultra centrifugal filter tubes (Merck-Millipore, Burlington, MA, USA).…”