Development of a Bio-Layer Interferometry-Based Protease Assay Using HIV-1 Protease as a Model

Abstract

Proteolytic enzymes have great significance in medicine and the pharmaceutical industry and are applied in multiple fields of life sciences. Therefore, cost-efficient, reliable and sensitive real-time monitoring methods are highly desirable to measure protease activity. In this paper, we describe the development of a new experimental approach for investigation of proteolytic enzymes. The method was designed by the combination of recombinant fusion protein substrates and bio-layer interferometry (BLI). The protease (PR) of human immunodeficiency virus type 1 (HIV-1) was applied as model enzyme to set up and test the method. The principle of the assay is that the recombinant protein substrates immobilized to the surface of biosensor are specifically cleaved by the PR, and the substrate processing can be followed by measuring change in the layer thickness by optical measurement. We successfully used this method to detect the HIV-1 PR activity in real time, and the initial rate of the signal decrease was found to be proportional to the enzyme activity. Substrates representing wild-type and modified cleavage sites were designed to study HIV-1 PR's specificity, and the BLI-based measurements showed differential cleavage efficiency of the substrates, which was proven by enzyme kinetic measurements. We applied this BLI-based assay to experimentally confirm the existence of extended binding sites at the surface of HIV-1 PR. We found the measurements may be performed using lysates of cells expressing the fusion protein, without primary purification of the substrate. The designed BLI-based protease assay is high-throughput-compatible and enables real-time and small-volume measurements, thus providing a new and versatile approach to study proteolytic enzymes.

Keywords: BLItz; HIV-1; bio-layer interferometry; human immunodeficiency virus; protease; protease assay; recombinant fluorescent protein substrate.

Figure 1

The RFPs as substrates of HIV-1 PR. (a) Schematic representation of the RFPs. The substrates contain cleavage sites of tobacco etch virus (TEV) and HIV-1 PR. (b) Cleavage site sequences of HIV-1 PR representing wild-type and modified HIV-1 MA/CA cleavage sites are shown, the modified residues are underlined. Asterisks show the cleavage position. (c) Native PAGE analysis of uncleaved substrates (~72 kDa) and cleavage products (~26 kDa) after proteolysis by HIV-1 PR_wt. The proteins were visualized in the gel using blue-light transillumination. (d) Reducing SDS-PAGE analysis of uncleaved substrates and cleavage products after proteolysis by HIV-1 PR_wt. The SDS-PAGE was followed by in-gel renaturation of the separated proteins, which were then visualized using UV transillumination. The substrates and fluorescent tag-containing products are indicated by arrows and asterisks, respectively.

Figure 2

Principle of the BLI-based protease assay. (a) Schematic representation of the biosensors during the assay. (b) A representative sensogram shows the steps of the assay procedure. After setting up the baseline using Ni-NTA biosensors in tube-mode, the RFP substrates are loaded onto the sensors in drop-mode until saturation. Loading is subsequently followed by tube- and drop-mode washing steps. Cleavage reactions were performed using a catalytically active (HIV-1 PR_wt, purple) and inactive HIV-1 PR (HIV-1 PR_ΔNΔC, orange) enzyme. The same Ni-NTA biosensor was used for the parallel experiments, and the sensor was regenerated between the measurements at low pH (based on manufacturer’s instructions).

Figure 3

Study of the interaction of different divalent cations with His₆-MBP-VSQNY*PIVQ-mEYFP substrate. The kinetic parameters determined for the cations are shown in Table 2. Results of two experiments are represented for each cation. The grey lines show fitting of data to 1:1 binding model by BLItz Pro software.

Figure 4

Dependence of initial change in the bio-layer thickness on enzyme concentration. The proteolysis was performed at increasing concentration of HIV-1 PR_wt. The initial ∆nm/∆t values were determined based on the slopes (2–10 s after initiation of the reaction). Error bars represent SD (n = 2).

Figure 5

Determination of the inhibitory effect of atazanavir on HIV-1 PR activity. (a) Representative sensograms of the enzyme reactions performed by using atazanavir in 2 nM and 2 µM final concentration. (b) Comparison of obtained initial ∆nm/∆t values which were determined based on the slopes (2–10 s after initiation of the reaction). Error bars represent SD (n = 2). Representative signal curves are shown for all steps of the assay in Figure S3a.

Figure 6

Determination of substrate specificity of HIV-1 PR using P2-modified RFP substrates. (a) Representative sensogram of the enzyme reactions with different substrates wt-9res (VSQNY*PIVQ) mut-9res (VSQLY*PIVQ). (b) Comparison of obtained ∆nm/∆t values. The initial ∆nm/∆t values were determined based on the slopes (2–10 s after initiation of the reaction). ***: p = 0.0004. Error bars represent SD (n = 4). (c) Kinetic parameters determined previously for HIV-1 PR using oligopeptide substrates representing 9-residue-long cleavage sites [44]. Representative signal curves are shown for all steps of the assay in Figure S3b.

Figure 7

Examination of the substrate groove of HIV-1 PR. (a) A representative sensogram of the enzyme reactions performed with wt-24res and mut-24res substrates. (b) Comparison of obtained ∆nm/∆t values. The initial ∆nm/∆t values were determined based on the slopes (2–10 s after initiation of the reaction). *: p = 0.0463. Error bars represent SD (n = 2). Representative signal curves are shown for all steps of the assay in Figure S3c.

Figure 8

SDS-PAGE analysis of the lysates of non-transformed (RFP−) and transformed (RFP+) BL21(DE3) cells. Arrow shows His₆-MBP-VSQNYPIVQ-mEYFP protein substrate (~72 kDa) detected in the cell lysates of transformed cells. Before UV transillumination, the proteins were renatured in the gel after separation by reducing SDS-PAGE (using 14% gel), then the gel was stained with Coomassie dye.

Figure 9

Cleavage reaction performed with non-purified RFP substrate on BLItz instrument. On a Ni-NTA biosensor, a baseline was initially established (tube-mode), then the sensor was merged into the cell lysate either containing (RFP+) or lacking (RFP−) the His₆-MBP-VSQNY*PIVQ-mEYFP substrate, until saturation were reached (drop-mode). Three individual washing steps were added to remove non-specifically bound proteins (tube-mode). Finally, HIV-1 PR_wt was added to the reactions (drop-mode) and the signal change was analyzed.