Purification and Biochemical Characterization of a New Protease Inhibitor from Conyza dioscoridis with Antimicrobial, Antifungal and Cytotoxic Effects

Abstract

The main objective of the current study was the extraction, purification, and biochemical characterization of a protein protease inhibitor from Conyzadioscoridis. Antimicrobial potential and cytotoxic effects were also examined. The protease inhibitor was extracted in 0.1 M phosphate buffer (pH 6-7). Then, the protease inhibitor, named PDInhibitor, was purified using ammonium sulfate precipitation followed by filtration through a Sephadex G-50 column and had an apparent molecular weight of 25 kDa. The N-terminal sequence of PDInhibitor showed a high level of identity with those of the Kunitz family. PDInhibitor was found to be active at pH values ranging from 5.0 to 11.0, with maximal activity at pH 9.0. It was also fully active at 50 °C and maintained 90% of its stability at over 55 °C. The thermostability of the PDInhibitor was clearly enhanced by CaCl₂ and sorbitol, whereas the presence of Ca²⁺ and Zn²⁺ ions, Sodium taurodeoxycholate (NaTDC), Sodium dodecyl sulfate (SDS), Dithiothreitol (DTT), and β-ME dramatically improved the inhibitory activity. A remarkable affinity of the protease inhibitor with available important therapeutic proteases (elastase and trypsin) was observed. PDInhibitor also acted as a potent inhibitor of commercial proteases from Aspergillus oryzae and of Proteinase K. The inhibitor displayed potent antimicrobial activity against gram+ and gram- bacteria and against fungal strains. Interestingly, PDInhibitor affected several human cancer cell lines, namely HCT-116, MDA-MB-231, and Lovo. Thus, it can be considered a potentially powerful therapeutic agent.

Keywords: Conyza dioscoridis; characterization antimicrobial effect; protease inhibitor; protein therapeutics.

Figure 1

Purification of PDInhibitor. (A) Gel filtration chromatography using a Sephadex G-50 column. A G-50 column (2 × 100 cm) was equilibrated with 0.1 Μ Tris-HCL buffer (pH 8) containing 0.2 Μ NaCl. The column was eluted at 4 °C with the same solution at a flow rate of 30 mL/hand, and 4.0 mL fractions were collected. (B) SDS-PAGE for Conyza dioscoridis protease inhibitor. 1: molecular markers (14 kD, 20 kD, 30 kD, 43 kD, 67 kD and 97 kD); 2: Sephadex G-50 PDInhibitor. (C) A reversed-phase analytical HPLC on C-8 column. A reverse phase RP-HPLC eurospher 100, C-8 column (250 × 4.6 mm), was equilibrated with 0.1% TFA in water. Protein elution was performed with an acetonitrile linear gradient (0–100%) at a flow rate of 1 mL/min over 60 min. 20µL of PDInhibitor (1 mg/mL) was used.

Figure 2

N-terminal sequence analysis of PDInhibitor from C. dioscoridis: Identical amino acids are in bold, and different amino acids are in boxes.

Figure 3

Effect of pH and temperature on PDInhibitor activity: Effect of pH on PDInhibitor activity (a) and stability (b). Inhibitor activity was assayed against trypsin at various pH values. For stability studies, the protease inhibitor was incubated in medium with different pH values for 12 h and assayed for residual inhibitor activity under standard conditions. Effect of temperature on PDInhibitor activity (c) and stability (d). Inhibitor activity was assayed against trypsin at various temperatures. For stability studies, the protease inhibitor was incubated at different temperatures, drawn at various time intervals and assayed for residual inhibitor activity under optimal pH and temperature conditions. The data shown are the mean ± SD (n = 3).

Figure 4

Effect of stabilizers on PDInhibitor activity at 70 °C. The inhibitor activity was determined under standard assay conditions (pH 7 and 37 °C) after 4 h of incubation of the inhibitor protein with various thermo stabilizers. The data are the means of triplicate determinations ± SD (n = 3).

Figure 5

Effects of metal ions at different concentrations on PDInhibitor activity. The protease inhibitor assay was performed at 37 °C and pH 7. The control represents 100% of the protease inhibitor activity under the same conditions in the absence of any metal. The data shown are the mean ± SD (n = 3).

Figure 6

Inhibitory activity of PDInhibitor toward several proteases with pharmaceutical and commercial importance. Data are the means of triplicate determinations ± SD.

Figure 7

(a): Antibacterial properties of the purified protease inhibitor PDInhibitor. The antibacterial properties of PDInhibitor were evaluated against several gram-positive and gram-negative bacteria. The bactericidal effect was assessed by measuring the protease inhibitor concentration necessary to kill 50% of the initial inoculum (IC50%), which was deduced from curves obtained from three independent experiments. Ampicillin was used as the positive reference standard, and acetate buffer was used as the negative control. (b): Antifungal properties of the purified protease inhibitor PDInhibitor. The antifungal properties of PDInhibitor were evaluated against several fungal strains. The fungicidal effect was assessed by measuring the protease inhibitor concentration necessary to kill 50% of the initial inoculum (IC50%), which was deduced from curves obtained from three independent experiments. Cycloheximide was used as the positive reference standard, and acetate buffer was used as the negative control.

Figure 7

(a): Antibacterial properties of the purified protease inhibitor PDInhibitor. The antibacterial properties of PDInhibitor were evaluated against several gram-positive and gram-negative bacteria. The bactericidal effect was assessed by measuring the protease inhibitor concentration necessary to kill 50% of the initial inoculum (IC50%), which was deduced from curves obtained from three independent experiments. Ampicillin was used as the positive reference standard, and acetate buffer was used as the negative control. (b): Antifungal properties of the purified protease inhibitor PDInhibitor. The antifungal properties of PDInhibitor were evaluated against several fungal strains. The fungicidal effect was assessed by measuring the protease inhibitor concentration necessary to kill 50% of the initial inoculum (IC50%), which was deduced from curves obtained from three independent experiments. Cycloheximide was used as the positive reference standard, and acetate buffer was used as the negative control.

Figure 8

Cytotoxic potency of PDInhibitor in Lovo (a), HCT-116 (b), and MDA-MB-231 (c) cells. Cytotoxicity was assessed using MTT assays by incubating cells for 24 h with various concentrations (25, 50, 100, and 200 μg) of crude inhibitor from C. dioscoridis (light gray bars), purified inhibitor PDInhibitor (dark gray bars) and pure protease inhibitor from Rhamnus Frangula (black bars), (d): Cell viability: HUVEC cells were maintained in culture for 72 h with (25–200µg/mL) concentrations of PDInhibitor using MTT solution.