High-level expression and molecular characterization of a recombinant prolidase from Escherichia coli NovaBlue

Abstract

Long-term use of organophosphorus (OP) compounds has become an increasing global problem and a major threat to sustainability and human health. Prolidase is a proline-specific metallopeptidase that can offer an efficient option for the degradation of OP compounds. In this study, a full-length gene from Escherichia coli NovaBlue encoding a prolidase (EcPepQ) was amplified and cloned into the commercially-available vector pQE-30 to yield pQE-EcPepQ. The overexpressed enzyme was purified from the cell-free extract of isopropyl thio-β-D-galactoside IPTG-induced E. coli M15 (pQE-EcPepQ) cells by nickel-chelate chromatography. The molecular mass of EcPepQ was determined to be about 57 kDa by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the result of size-exclusion chromatography demonstrated that the enzyme was mainly present in 25 mM Tris-HCl buffer (pH 8.0) as a dimeric form. The optimal conditions for EcPepQ activity were 60 °C, pH 8.0, and 0.1 mM Mn²⁺ ion. Kinetic analysis with Ala-Pro as the substrate showed that the K _m and k _cat values of EcPepQ were 8.8 mM and 926.5 ± 2.0 s^-1, respectively. The thermal unfolding of EcPepQ followed a two-state process with one well-defined unfolding transition of 64.2 °C. Analysis of guanidine hydrochloride (GdnHCl)-induced denaturation by tryptophan emission fluorescence spectroscopy revealed that the enzyme had a [GdnHCl]_0.5,N-U value of 1.98 M. The purified enzyme also exhibited some degree of tolerance to various water/organic co-solvents. Isopropanol and tetrahydrofuran were very detrimental to the enzymatic activity of EcPepQ; however, other more hydrophilic co-solvents, such as formamide, methanol, and ethylene glycol, were better tolerated. Eventually, the non-negative influence of some co-solvents on both catalytic activity and structural stability of EcPepQ allows to adjust the reaction conditions more suitable for EcPepQ-catalyzed bioprocess.

Keywords: Chaotropic agent-induced denaturation; Escherichia coli; Gene expression; Molecular characterization; Organic co-solvents; Prolidase.

Figure 1. Analyses of the soluble proteins and specific activity of E. coli M15 (pQE-EcPepQ) under a specific condition.

(A) Schematic diagram of the key elements of pQE-EcPepQ. (B) Analysis of the crude extracts by SDS–PAGE. Lanes: M, protein size markers; 1, cell growth without IPTG induction; 2, cell growth with 5 μM IPTG induction; 3, cell growth with 10 μM IPTG induction; 4, cell growth with 50 μM IPTG induction; 5, cell growth with 100 μM IPTG induction; 6, cell growth with 500 μM IPTG induction. (C) Effects of incubation temperature and IPTG concentration on the production of active EcPepQ. The amount of active enzyme was determined by measuring the prolidase activity of the soluble extracts shown in (B). These data were a representative of three independent experiments.

Figure 2. Gel electrophoresis and size-exclusion chromatography of the recombinant enzyme.

(A) SDS–PAGE analysis. Lanes: M, protein size markers; 1, the crude extract of E. coli M15 (pQE-EcPepQ); 2, the enzyme sample after Ni-NTA purification. (B) FPLC analysis. Blue dextran 2000 was used to determine the void volume. The K_av values for the standard proteins and EcPepQ were plotted against the logarithm of their molecular weights to estimate the native molecular mass of EcPepQ. (C) Native PAGE analysis. Lanes: M, protein size markers; 1, the enzyme sample after Ni-NTA purification.

Figure 3. Effects of temperature and pH on activity (A and B) and stability (C and D) of EcPepQ.

Enzyme assay was performed as aforementioned procedures with one mM Ala-Pro as the substrate. 100% relative activity refers to the percentage of the initial reaction rate obtained by the enzyme at pH 8.0 and 60 °C. The residual activity was expressed as a percentage of specific activity with the untreated sample being defined as 100%. The data are expressed as mean ± SD of three independent experiments.

Figure 4. Effects of divalent metals (A) and different concentrations of Mn²⁺ ion (B) on the catalytic activity of EcPepQ.

In these experiments, the sample without extrinsic metal ions was used as a control and the EDTA-treated enzyme served as a negative reference. The data were expressed as mean ± SD of three independent experiments.

Figure 5. Far-UV CD and intrinsic tryptophan fluorescence spectra of EcPepQ.

(A) Temperature-dependent CD spectra of the enzyme. Far-UV CD spectra of the enzyme were recorded at the indicated temperatures over the wavelength range of 190–260 nm. (B) Transition and cooling curves of the enzyme . The temperature-induced unfolding of the enzyme was monitored at three different heating rates as aforementioned procedures. The blue line represents the cooling curve of the unfolded protein, which had been heated with a scan rate of 2.0 °C/min. (C) Temperature-dependent fluorescence spectra of the enzyme. Fluorescence spectra of the enzyme were recorded at the indicated temperatures over the emission wavelength range of 305–500 nm.

Figure 6. Concentration effect of GdnHCl on the catalytic activity of EcPepQ (A) and the corresponding changes in the tertiary structure as monitored by AEW value (B).

The purified enzyme at a final concentration of 0.15 mg/mL was incubated with different concentrations of GdnHCl at 30 °C for 10 min. Then, the sample solutions were subjected to measurement of prolidase activity under the standard assay conditions and fluorescence analysis.

Figure 7. Effect of different water-miscible organic co-solvents on the enzymatic activity of EcPepQ.

The enzymatic activity was determined in the reaction mixture with different concentrations of organic co-solvents under the standard assay conditions. The residual activity was expressed as a percentage of specific activity with the solvent-free sample being defined as 100%.

Figure 8. Far-UV CD (A) and intrinsic tryptophan fluorescence (B) spectra of EcPepQ in the presence of water-miscible organic co-solvents.

Spectral analyses of the enzyme were carried out at 25 °C in either 25 mM Tris–HCl buffer (pH 8.0) or the buffer supplemented with various organic co-solvents at concentrations that led to reductions in the catalytic activity of ≥90%.