Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing (IEF) Method

Summary

The objective is to fractionate and isolate bioactive small molecules, peptides from a complex plant extract, and proteins from pathogenic microbes by employing liquid-phase isoelectric focusing (IEF) method. Further, the separated molecules were identified and their bioactivity confirmed.

Abstract

Natural products derived from plants and microbes are a rich source of bioactive molecules. Prior to their use, the active molecules from complex extracts must be purified for downstream applications. There are various chromatographic methods available for this purpose yet not all labs can afford high performance methods and isolation from complex biological samples can be difficult. Here we demonstrate that preparative liquid-phase isoelectric focusing (IEF) can separate molecules, including small molecules and peptides from complex plant extracts, based on their isoelectric points (pI). We have used the method for complex biological sample fractionation and characterization. As a proof of concept, we fractionated a Gymnema sylvestre plant extract, isolating a family of terpenoid saponin small molecules and a peptide. We also demonstrated effective microbial protein separation using the Candida albicans fungus as a model system.

Introduction

The purification of biomolecules from complex biological samples is an essential and often difficult step in biological experiments¹. Isoelectric focusing (IEF) is well-suited for high resolution separation of complex biomolecules where carrier ampholytes travel according to their charge and establish the pH gradient in an electric field³. The first commercial carrier ampholyte for IEF was developed by Olof Vesterberg in 1964 and patented^4,5. Carrier ampholytes are aliphatic oligo-amino oligo-carboxylic acid molecules of varying length and branching⁶. Subsequently, Vesterberg and others improved the carrier ampholytes for their expanded use in separating biomolecules^6,7.

Methods to separate biomolecules include agarose and polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis (2-DE), isoelectric focusing, capillary electrophoresis, isotachophoresis and other chromatographic techniques (e.g., TLC, FPLC, HPLC)². Liquid-phase IEF performed in an instrument called a “Rotofor” was invented by Milan Bier⁸. He pioneered the concept and design of this instrument and contributed extensively to the theory of electrophoretic migration. His team also developed a mathematical model of electrophoretic separation process for computer simulations⁹.

The liquid-phase IEF apparatus is a horizontally rotating cylindrical cell consisting of a nylon core divided into 20 porous compartments and a circulating water cooling ceramic rod. The porous chambers allow molecules to migrate through the aqueous phase between the electrodes and permit collection of purified samples under vacuum in fractions. This purification system can provide up to 1000-fold purification of a specific molecule in <4 hours. A valuable feature of this instrument is that it can be applied as a first step for purification from a complex mixture or as a final step to achieve purity¹⁰. If the molecule of interest is a protein, another advantage is that its native conformation will be maintained during the separation.

The use of liquid-phase IEF has been reported widely for proteins, enzymes and antibody purification^{6,10,11,12,13,14}. Here we describe the use of this approach for separating and purifying small molecules and peptides from the medicinal plant Gymnema sylvestre. This protocol will help researchers concentrate and purify active small molecules from a plant extract for downstream applications at low cost. In addition, we also demonstrate that enrichment of proteins from a complex protein extract from Candida albicans fungus¹⁵ in this IEF-based system as a second example.

Protocol

1. Setup and prerunning of standard liquid-phase IEF unit

1. Assemble the liquid-phase IEF electrodes (anode-red button and cathode-black button) with their respective exchange membranes according to the instruction manual (see Table of Materials). Equilibrate the anion exchange membranes with 0.1 M NaOH and the cation exchange membranes with 0.1 M H₃PO₄ at least for 16 h when new membranes are used.
   1. Store the membranes in electrolytes (0.1 M NaOH or 0.1 M H₃PO₄) between runs and do not allow to dry out.
2. Assemble the inner and outer portion of the electrode by aligning three oblong holes in the ion-exchange gaskets. Fill the electrodes with respective electrolytes (~25-30 mL) to prevent their membranes from drying out.
   1. Do not add excess (more than 1/3^rd of electrode chamber volume) electrolyte that may build up pressure inside the electrode and cause leakage.
3. Cover the sample collection ports with sealing tape that comes with the instrument assembly parts (see Table of Materials). The sample collection ports side can be identified by the two vertical metal aligning pins. Alternatively, use standard sealing tape to seal the ports.
4. Assemble all parts of the focusing chamber assembly in sequence (anode electrode, nylon membrane core, focusing chamber and cathode electrode) over the ceramic cooling finger.
5. Fill the focusing chamber with precooled distilled water (total volume of 60 mL for the standard IEF cell) using a 50 mL syringe.
6. Connect the liquid-phase IEF instrument to a circulating cooling water at 4 ºC and prerun the unit at 15 W and 3,000 V for 3-5 min or until the voltage stabilizes.
   NOTE: Generally, within one minute, the voltage will reach the maximum set values. Prerunning with distilled water helps to remove the residual ions from the focusing chamber and the nylon membrane core.
7. Switch off the power source and remove the water from the cell using the fraction collector. Re-seal the collection ports with sealing tape.
   NOTE: Now the instrument is ready for use in step 2.4.

2. Separation and purification of small molecules and peptides from Gymnema sylvestre  extract

1. Measure 0.6 g of plant extract and dissolve in distilled water (60 mL) by mixing in a roller tube for 5 min.
   NOTE: Any biological sample that is soluble and free of salt can be used for separation and purification using this IEF instrument. Samples with buffering salt concentration up to 10 mM can be used with slightly decreased resolution. We are interested in the saponin family of triterpenoid compounds, the gymnemic acids, from G. sylvestre plant for their unique antifungal properties¹⁶.
2. Centrifuge the solubilized plant extract at 10,000 x g for 5 min to remove insoluble particles.
3. Transfer the supernatant (~60 mL) to an 80 mL centrifuge tube, and mix it with 0.6 mL of ampholyte (pH 3-10) to 1% (v/v).
4. Follow steps 1.1 – 1.7 to prepare the liquid-phase IEF unit. The chamber is now ready to load the sample.
5. Use a 50 mL syringe with a 1-1/2 inch 19 G blunt end needle (comes with the instrument) and load the prepared sample with ampholyte (60 mL total) in the cell through sample collection ports.
6. Remove air bubbles from the sample cell by removing the focusing cell from the stand and tapping the electrode chamber to dislodge the bubbles. The presence of air bubbles will cause voltage and current fluctuations and affect the run.
7. Connect the unit to the water coolant (4 °C) and start fractionation with the power supply at a constant 15 W.
8. Run the apparatus for 3 h or until the voltage reaches a constant value.
   NOTE: As the sample starts focusing, the voltage will start climbing gradually until it reaches a constant value.
9. After the run, prepare to collect the fractions in the harvest box (containing 20 plastic tubes, 12 mm x 75 mm, 5-6 mL volume) connected to the vacuum pump by pressing the harvest button ON in the IEF unit.
10. Align the 20-collection pins with the 20-collection ports of the focusing cell that is sealed with the tape.
11. Push the collection pins through the sealing tape and turn the vacuum pump ON simultaneously (see Figure 2B, 2C & 2D).
    NOTE: About 3 mL fractions from each chamber will be collected into the tubes for each time. Fractions can be used for subsequent downstream applications (SDS-PAGE for peptide quantification, TLC and bioassay for small molecules).

3. Separation and purification of proteins from  C. albicans

1. Grow single colony of C. albicans in yeast-peptone-dextrose (YPD) broth¹⁶ at 30 °C with shaking (200 rpm) for overnight.
2. Collect the yeast cells by centrifugation (10,000 x g for 5 min).
3. Suspend C. albicans yeast cells in ammonium carbonate (1.89 g/L) buffer containing 1% (v/v) beta-mercaptoethanol (β-ME) (1/10th of the culture volume) and rotate in a roller tube for 1 h at 5 °C¹⁵.
4. Remove the yeast cells by centrifugation (10,000 x g for 5 min) and filter the protein extract through a 0.45 µm filter.
   NOTE: Protein samples from C. albicans cytosol, membrane or cell wall can be prepared and used for IEF fractionation. Similarly, proteins from bacteria or other biological samples (animal tissue extract) can be used after removing any salt by appropriate methods (e.g., by dialysis or using desalting columns).
5. Dialyze the protein extract in a dialysis tubing (3,500 MWCO) against water for 15 h at 4 °C. Estimate the protein concentration by the Bradford dye-binding method using gamma globulin as a standard¹⁷.
6. Use 500 mg of total protein in 60 mL of water containing 1% (v/v) ampholyte (pH 5-8) for the fractionation.
   NOTE: Since a broad-range ampholyte (pH 3-10) does not enrich certain non-glucan attached cell wall proteins of C. albicans well, use a narrow-range (pH 5-8) ampholyte. An ampholyte concentration of up to 2% can be used if the sample protein concentration is more than 2 mg/mL; this minimizes protein aggregation during focusing. Always keep the samples, ampholytes, and water precooled on ice.
7. Repeat steps 1.1- 1.7 to prepare the IEF unit and use the protein solution from step 3.6 to load into the IEF cell.
8. After 4 h of focusing at a constant 15 W, harvest protein fractions (1-20) as described above (steps 2.9-2.11) and analyze on 12.5% SDS-PAGE after reducing and boiling the protein samples¹⁸.
9. Stain the resolved proteins by staining the SDS-PAGE gel with the Coomassie blue dye (0.01%) solution for 2-3 h at room temperature on a rocker. Destain the gel and record the gel image using a gel imager.
   NOTE: Coomassie blue dye (0.01%) can be prepared by mixing 0.01 g of Coomassie blue powder in a destaining solution containing 40% methanol and 10% acetic acid.

4. Bioactivity of purified small molecules from plant extract Gymnema sylvestre

1. Grow C. albicans in yeast cells as in step 3.1.
2. To prepare cell suspension, dilute overnight culture of C. albicans yeast cells (1/1000 dilution) into a fresh RPMI cell culture medium supplemented with 50 mM glucose.
   NOTE: C. albicans converts from yeast growth to hyphae under hyphal inducing conditions (RPMI at 37 °C). Gymnemic acid small molecules are shown to inhibit the conversion of yeast cells into hyphae under hypha inducing conditions¹⁶. We aimed to determine if liquid-phase-IEF-separated G. sylvestre extract contain these bioactive molecules.
3. From the prepared cell suspension, add 90 μL to each well of the 96-well plate.
4. From each fraction (1-20 harvested fractions of G. sylvestre extract obtained from liquid-phase IEF, Figure 4), add 10 μL into the wells with 90 μL of the above cell suspension (step 4.2). Perform the assay in triplicate.
5. Add 10 μL of water that contains ampholyte (1%) into separate wells with 90 μL of C. albicans yeast cell suspension as a negative control.
   NOTE: Ampholytes have bioactivity potential and so it is essential to include an ampholyte control while performing any bioassay¹⁹.
6. Incubate the 96-well plate at 37 °C for 12 h and observe the inhibition of C. albicans yeast-to-hypha conversion under the microscope¹⁶. Also, determine the percentage of inhibition of yeast-to-hypha conversion.

Results

Separation and purification of small molecules and peptides from Gymnema sylvestre plant extract
Using the preparative liquid-phase IEF method, we fractionated medicinal plant extracts and cell surface proteins from a human pathogenic fungus, C. albicans. A schematic of these fractionation protocols is shown in Figure 1.

From 20 fractions of G. sylvestre extract obtained from liquid-phase IEF, the dark-colored mole...

Discussion

Small molecules from natural product sources (e.g., plants) include complex secondary metabolites that are highly diverse in chemical structure. They are believed to be involved in plant defense mechanisms. In addition, polypeptides are also present in plant tissues²². These natural product small molecules are rich sources of test molecules for drug discovery and development. However, the difficult and tedious methods required for their isolation and purification limit their use for therapeutic ap...

Materials

Name	Company	Catalog Number	Comments
0.45 µm syringe filter	Fisher scientfic	09-720-004
2-Mercaptoethanol	Sigma	M3148
Ammonium carbonate	Sigma-Aldrich	207861-500
Bio-Lyte 3/10 Ampholyte	Bio-Rad	163-1113
Bio-Lyte 5/8 Ampholyte	Bio-Rad	163-1192
Compact low temperature thermostat	Lauda -Brinkmann	RM 6T	Set water cooling to 4 oC and it can be run even at 0 oC as when it passes through the Rotofor cooling core, the circulating water temperature will be around 5 or more depending on the voltage.
Coomassie Brilliant Blue R	Sigma-Aldrich	B7920
Dialysis tubing (3,500 MWCO)	Spectrum Spectra/Por	132112T
Gymnema plant leaf extract powder (>25% Gymnemic acids)	Suan Farma, NJ USA
Incubator	Lab companion	SI 300R
Microscope	Leica	DM 6B
Mini protean electrophoresis	Bio-Rad
pH meter	Mettler Toledo	S20	Useful to determine the pH of the Rotofor (liquid-phase IEF) fractions
Rotofor	Bio-Rad	170-2972	http://www.bio-rad.com/webroot/web/pdf/lsr/literature/M1702950E.pdf (Rotofor Instruction manual for assembling the unit)
RPMI-1640 Medium	HyClone	DH30255.01
Sealing tape	Bio-Rad	170-2960	Scotch tape may also be used.
Sorvall legend micro 17 centrifuge	Thermo scientific	75002432
TPP tissue culture plate -96 well flat bottom	TPP	TP92696