Figure 1.
Schematic overview of the fluorescence-assisted whole-cell assay for characterization of proteases.
(A) Short-lived fluorescent reporter substrates (GFP-PS-ssrA) and a protease of interest are coexpressed in E.coli. Protease-mediated removal of the ssrA-tag from the reporter construct, through in vivo processing of the substrate peptide (PS), rescues GFP from cytoplasmic degradation. This enhances the fluorescence intensity of the entire cell, thus enabling flow cytometry analysis and cell sorting to collect desired clones. Explanations: GFP, green fluorescent protein; PS, protease substrate peptide; ssrA, a ClpXP-specific degradation tag. (B) Flow cytometry analysis of E. coli cells that coexpress TEVp and different reporters constructs, 2.5 h after induction (0.1 mM IPTG, 0.2% arabinose). Three reporter constructs contained closely related TEVp substrate sequences that only differed in the P1′ position. They are represented by the histograms shown in green (DH5α/pMal-TEV2/pGFP-subG-ssrANY; subG = ENLYFQG), purple (DH5α/pMal-TEV2/pGFP-subV-ssrANY; subV = ENLYFQV) and blue (DH5α/pMal-TEV2/pGFP-subP-ssrANY; subP = ENLYFQP). The negative control (DH5α/pMal-TEV2/pGFP-ssrANY) and the positive control (DH5α/pMal-TEV2/pGFP-subG) are shown in red and dark green, respectively.
Table 1.
Incidence and in vivo processing efficiency of peptides that emerged from substrate library screening.
Figure 2.
Screening of TEVp substrate libraries.
Three different combinatorial substrate libraries were created, using NNK degenerate codons for randomization of position P6, P3, P1 (Lib1); P6, P3, P1, P1′ (Lib2); or P5, P4, P2, P1′ (Lib3) within the cognate TEVp substrate peptide (ENLYFQG). The libraries were then screened for optimal TEVp substrates. (A) Pre-sorting procedure to eliminate false positive clones from the libraries: DH5α/pMal-TEV2/pGFP-Lib1-ssrANY, DH5α/pMal-TEV2/pGFP-Lib2-ssrANY or DH5α/pMal-TEV2/pGFP-Lib3-ssrANY cells expressing the substrate libraries alone (i.e., TEVp expression not induced) were analyzed on a flow cytometer and non-fluorescent cells were collected through sorting. Here, the original non-sorted population from library 2 and the corresponding “false-positive depleted” library (after two rounds of sorting) are represented by the histograms in purple and jade, respectively. Library 1 and 3 exhibited the same appearance as library 2 (data not shown). (B) Enrichment-progress when screening the libraries for functional TEVp substrates: The false-positive depleted libraries (see Figure 2A), now coexpressing TEVp and the substrate libraries, were subjected to quantitative flow cytometry analysis, and highly fluorescent cells were collected. The populations from the false-positive depleted library 2, before (jade), after the first (black) and second round of sorting (green) are shown. All three libraries had similar appearance (data not shown).
Figure 3.
Flow cytometry analysis of individual clones that emerged through screening of library 1 and 2.
Each histogram corresponds to a pure culture of DH5α cells coexpressing TEVp and a reporter construct containing a unique substrate peptide, 2.5 h after induction (0.1 mM IPTG, 0.2% arabinose). The different peptides analyzed are indicated in the figure.
Figure 4.
Substrate conversion of different ABP-PS-ZZ fusion proteins by recombinantly produced TEVp.
(A) SDS-PAGE analysis (representative of three independent experiments). ABP-PS-ZZ (10 µg) was incubated with TEVp (0.3 µg) in TEVp reaction buffer at 37°C for different incubation times (0, 5 min, 15 min, 30 min, 45 min, 60 min, 90 min, 2 h, 3 h, 4 h, 5 h and 6 h). The substrate peptide sequence (PS) in each analyzed fusion protein is indicated in the figure. As a negative control, we used a fusion protein containing a peptide (SNLVFGP) that cannot be cleaved by TEVp. (B) The substrate conversion of each given fusion protein plotted against time based on densitometric analysis of the SDS-PAGE gels in Figure 4A.