Team:Lethbridge/Protocols

Introduction

The peptide purification team prepares, extracts, and characterizes the peptides being expressed in both E. coli and potatoes. Our strategy involves first identifying a variety of potential antimicrobial peptides. The peptides we chose to research came from a range of different organisms: some were native peptides to the potato, but some came from other organisms, such as BMAP-18 from cows. Secondly, we looked at the properties of these peptides by reading through different research papers that examined each peptide. This also allowed us to assess whether the peptides could be harmful to humans, and such peptides were eliminated. After this, our third task is to develop an optimal purification method based on our research of current methods, and protocols for the preparation, extraction and characterization of our chosen peptides. For our experimentation, we collaborated with Mariana Vetrici from Dmytro Yetvushenko’s laboratory working with the BMAP-18 peptide. Firstly, the gene was cloned, transgenic plants were propagated, grown in tissue culture and transferred to the greenhouse for tuber production. Total proteins were extracted from tubers and results were visualized by SDS PAGE and Coomassie staining.

Protein Extraction Protocols

One of the main things that the peptide purification team is concerned with is extracting protein from potatoes. Below are three extraction methods that we found to isolate total potato protein. We have followed the first method for our extraction, but the phenol and SDS are available extraction methods in case we want to optimize the amount of BMAP-18 in future experiments.

Protein Isolation Method from Yevtushenko's lab courtesy of Mariana Vetrici

Tissue was weighed and ground in liquid nitrogen. The powder was suspended in 3 mL extraction buffer per 1 mg tissue, in 1.5 ml Eppendorf tubes. Extraction buffer: 65 mM tris(hydroxymethyl)aminoethane (Tris) (pH 6.8), 1% sodium dodecyl sulfate (SDS), 5% glycerol, 2.5% EtSH. The suspension was set in a heating block preheated to 100 °C for 5 min. The tubes were immediately frozen in liquid nitrogen for 5 min, and then placed again in the 100 °C heating block for an additional 5 min. The samples were centrifuged at 16,000 x g and 4 °C for 25 min. The supernatant, containing total cellular proteins, was removed to a clean, sterile Eppendorf tube. Protein concentration was determined via the Bradford assay (Bradford, 1976). A standard curve was prepared with BSA diluted over a range of 2 – 9 μg/mL. To determine protein concentration as accurately as possible and to minimize interference by SDS, 10 μl aliquots of total cellular protein extracts were assayed in 1 ml reactions, thus reducing the SDS concentration to 0.01%.

Phenol extraction and SDS extraction (Following Pierre Delaplace, et al., 2006)

Protocols that could be used for extraction of potato protein to test for BMAP-18 presence.

Phenol extraction

Extraction Buffer: 0.7 M sucrose, 50mM EDTA, 0.1 M KCl, 10 mM thiourea, 0.5 M Tris pH 7.5, 2 mM PMSF, 50 mM DTT. (~450 mL)

Rehydration Buffer: 5 M urea, 2M thiourea, 2% w/v CHAPS, 2% w/v 3-(4-heptyl)phenyl-3-hydroxy-propyl-dimethylammonio-pro-panesulfonate (C7BzO), 20 mM DTT, 5 mM tris(2-carbosy-ethyl) phosphine hydrochloride (TCEP-HCl). (~30 mL)

  1. 1g of tissue is mixed with 4mL of extraction buffer.
  2. Incubate the mixture in ice for 10 minutes.
  3. Centrifuge the mixture at 13 000 x g for 15 minutes at 4 °C.
  4. Mix supernatant with 5 mL of phenol (pH 8.0).
  5. Vortex mixture for 10 minutes (at room temperature).
  6. Centrifuge for 10 minutes at 6000 x g (at room temperature).
  7. Combine 5 mL of extraction buffer to the phenol mixture.
  8. Vortex mixture at 1800 rpm for 10 minutes (at room temperature).
  9. Centrifuge mixture for 10 minutes at 6000 x g (at room temperature).
  10. Remove the aqueous layer.
  11. Combine the proteins with 0.1M ammonium acetate in methanol for 20 mL.
  12. Centrifuge mixture for 20 minutes at 20 000 x g at 4 °C.
  13. Rinse the pellet with 4 mL 0.1M ammonium acetate in menthol and repeat one more time.
  14. Rinse the pellet with 10 mM DTT cold acetone.
  15. Air-dry the pellet for 30 minutes.
  16. Dissolve the pellet in 600 μL of rehydration buffer at 45 minutes (at room temperature).
  17. Aliquot the mixture.
  18. Store the mixture at - 80 °C.
SDS extraction

SDS Lysis Buffer: (4% w/v SDS, 5% w/v sucrose, 10% w/v poly(polyvinylpolypyrrolidone), 0.3 % w/v DTT, 20 mM sodium phosphate pH 7.0).

  1. Mix 1g of the potato tissue with 2 mL of hot SDS lysis buffer.
  2. Incubate the mixture for 10 minutes at 65 °C.
  3. Centrifuge mixture at 15000 x g for 15 minutes at 4 °C.
  4. Remove the residue.
  5. Centrifuge the supernatant at 15000 x g for 15 minutes.
  6. Remove the residue.
  7. Precipitate the proteins with 8mL 10mM of DTT cold acetone.
  8. Centrifuge mixture at 20000 x g for 20 minutes at 4 °C.
  9. Rinse the pellet with 10 mM cold acetone (repeat twice).
  10. Air-dry the pellet for 30 minutes.
  11. Rinse the pellet in 600 μL of rehydration buffer within 75 minutes (at room temperature.
  12. Aliquot the mixture.
  13. Store at -80 °C.

Cloning, Transgene Confirmation, Protein Extraction, and SDS PAGE Results for the BMAP-18 Peptide

All procedures were carried out in collaboration with Mariana Vetrici from Dmytro Yevtushenko’s lab.

Cloning Results from E. coli stocks

Two BMAP18 constructs were created using 2 versions of the PmBiP promoter. This promoter was chosen because it originated from plants and showed organ-specific expression, such as tubers and leaves. Furthermore, this promoter is wound-inducible allowing for upregulation of expression. In the first construct, BMAP18 was placed under control of the full-length PmBiP promoter (1-1PmBiPPro) and in the second construct, BMAP18 was under control of the truncated promoter 1-3PmBiPPro. The constructs were inserted in the pBI121 binary vector and cloned E. coli. Construct integrity was confirmed by restriction enzyme analysis and PCR, and products were visualized by agarose gel electrophoresis.

Figure 1. 1-1PmBiPPro/BMAP18 and 1-3PmBiPPro/BMAP18 constructs were confirmed by PCR analysis of E. coli stocks. The 2339 bp and 1339 bp regions of the inserted gene constructs corresponding to each of the two promoters and the BMAP18 gene were amplified by PCR with forward primers based on the 5’-sequence of the PmBiP Promoters and the reverse primer corresponding to the 3’-sequence of the BMAP-18 gene. The results were visualized on a 1% agarose gel. The length of the 1-1/BMAP-18 construct is 2339 bp and the 1-3/BMAP-18 construct is 1339 bp.

The vector was transferred from E. coli to Agrobacterium. Agrobacterium-mediated transformation was used to insert the transgene into potato plants of the Desiree cultivar. The plants were propagated in tissue culture.

Transgene Confirmation in Plants

Following the Agrobacterium-mediated transformation, potato plants were grown in tissue culture and the presence of the transgene was confirmed by PCR.

Figure 2. The presence of the BMAP-18 transgene under control of the 1-1PmBiPPro and 1-3PmBiPPro in potato plants was confirmed by PCR. PCR products were visualized on a 1% agarose gel. A, 1-1 PmBiPPro/BMAP-18 lines. B, 1-3 PmBiPPro/BMAP-18 lines. For PCR, the primers were based on the 5’ sequence of PmBiPPro1-1/PmBiPPro1-3 and the 3’ sequence of BMAP-18. 16 Lines with the 1-1/BMAP18 construct were produced. The lines are designated as “1” for the 1-1 promoter, and -# for the line. PCR was conducted to confirm the presence of the transgene. The expected size of the PCR product is 2339 bp.

Figure 3. The potato plants transformed with the BMAP-18 gene by Agrobacterium-mediated plant transformation. Plants that were initially grown in tissue culture were transferred to the greenhouse for producing tubers. The plants were healthy, flowered, and produced tubers.

Figure 4. Potato tubers from lines representing both promoters, with or without the transgene. 1-1-8 and 1-3-6 contain the BMAP-18 transgene, whereas 1-3-13 and 1-1-3 do not. These potato tubers were used for protein extraction and the wounding experiment.

Protein Extraction from Wounded and Unwounded Potato Tissue

For protein isolation, the protein was extracted directly from normal tissue and tissue subjected to mechanical wounding, which mimics pathogen invasion. Previous work showed that the PmBiP Promoters were wound inducible (Yevtushenko and Misra, 2018).

Figure 5. Coomassie stained SDS PAGE gels showing successful isolation of potato tuber proteins. The extraction protocol was followed for total protein extraction from potatoes and confirmed via SDS PAGE. The constructs all had a wound-inducible promoter PmBiPPro1-1 or PmBiPPro1-3, and those with the “W” designation were wounded. A, 18% SDS PAGE gel. B, 15% SDS PAGE gel.

SDS PAGE of Proteins

8 mL of 18% separating gel (2.2 mL ddH2O, 3.6mL 40% Acrylamide, 2mL of 1.5 M Tris pH 8.8, 80 µL 10% SDS, 80 µL 10% APS, 8 µL TEMED) was made for the first gel.

8 mL of 15% separating gel (2.8 mL ddH2O, 3mL 40% Acrylamide, 2 mL of 1.5 M Tris pH 8.8, 80 µL 10% SDS, 80 µL 10% APS, 8 µL TEMED) was made for the second gel.

Both gels used the same stacking gel recipe, where 5mL was made (2.9 mL ddH2O, 0.75 mL 40% Acrylamide, 1.25 mL of 1.5 M Tris pH 8.8, 50 µL 10% SDS, 50 µL 10% APS, 5 µL TEMED).

Sample Preparation for the 18% and 15% SDS PAGE Gels

Potato protein sample concentrations were measured using the Genesee BioDrop DUO - 85-717.

Laemmli Loading Buffer for sample preparation:

2.5 mL 2 M Tris-HCL (pH 6.8), 2 g sodium dodecyl sulfate, 100 mg bromophenol blue, 10 mL glycerol, 1.542 g DTT, Add up to 20 mL of dH2O. Generate 100-μL aliquots of the buffer, and store them at −20 °C for up to 3 months. Loaded 60 μg of protein based on concentration.

Coomassie Staining and Destaining

  1. Remove SDS-PAGE gel from glass and rinse once in ddH2O in a suitable container with a lid. Try not to use a container much larger or much smaller than the gel.
  2. Add enough Coomassie Stain to cover the gel by 1/2 inch (~ 1.5 cm).
  3. Microwave on high power for 40 seconds to 1 minute (until the Coomassie Stain boils).
  4. Incubate the gel in the Coomassie stain for 5 to 10 minutes on a rocking table. If you did not microwave the Coomassie/gel, incubate for at least 1 hour.
  5. Pour off the Coomassie Stain. The Coomassie Stain can be recycled a couple of times by filtering it.
  6. Rinse twice in ddH2O or used Destain solution to remove Coomassie Stain from the container.
  7. Add fresh Destain solution to cover the gel by 3/4 inch (~ 2 cm).
  8. Tie Kimwipes in a simple knot and place 4 of them in the Destain solution around the gel. Try to avoid laying the Kimwipes on the gel as this will cause an uneven destaining.
  9. Microwave on high power for 40 seconds to 1 minute (until the Destain boils).
  10. ncubate the gel in the Destain solution for 10 minutes on a rocking table. If you did not microwave the Coomassie/gel, incubate for at least 1 hour.
  11. Discard the stained Kimwipes and replace with fresh knotted Kimwipes.
  12. Incubate a second time for 10 minutes to overnight on a rocking table. Stop whenever the level of destaining is sufficient for you. Microwave again to speed up the process.
  13. The used Destain solution can be recycled a couple of times by storing it in a sealed container with sponges or Kimwipes to remove all traces of Coomassie Stain.

Peptide purification

Fast Protein Liquid Chromatography

FPLC was a method developed to provide high-resolution separation of biopolymers, including proteins, at a lower cost.

Figure 6. Schematic of the FPLC system.

We will detail the FPLC method based off of the paper from Madadlou et al, (2011):

Buffer A: 10mM tris-HCl, pH 7.0. 0.22 μm filter and degas.

Buffer B: 10mM Tris-HCl, pH 7.0, 0.1 M NaCl.0.22 μm filter and degas.

Buffer C: 50mM Tris-HCl, pH 7.0, 100mM KCl.

3.1 Sample preparation

Prepared with material that has previously been exposed to chromatography.

  1. Sephadex G-25 resin is identified through aspiration chromatography.
  2. Column is equilibrated by using column 3 to 4 volumes of buffer A.
  3. The conductivity is measured using a conductivity meter.
3.2 FPLC Modes

3.2.1 Ion Exchange FPLC

Ion-exchange chromatography separates ions and polar molecules based on their affinity to the ion exchanger.

  1. Prime P-900 pumps A and B with 0.22 μm filter and degas respectively.
  2. P-500 pump pressure limit set below max for column.
  3. Mono Q column equilibrated with 5X buffer A volume, and 10X buffer B volume, then another 5X volumes of buffer A.
  4. 0.5 mL to 10mL of sample is loaded, then washed and assayed for protein of interest.
  5. Repeat procedures if proteins do not bind to Mono Q column with a Mono S column.
  6. Develop column by gradually increasing concentration of buffer B Regenerate column by washing with 10X buffer B volume, followed by 5X volumes of buffer A.

3.2.2 Scouting FPLC method

  1. Create a gradient by varying the percent of strong solvent added, from which the percent where protein eluted is determined.
  2. To determine the best buffer, a chromatography using varying pH values using different buffer systems is carried out. Running a single sample at a number of different pH values on a column may separate two peptides that elute at similar pHs.
  3. The larger sample volume can be run further and analyzed, once optimal elution conditions are found.
3.3 Gel filtration FPLC

This step is often seen as a ‘polishing’ step.

  1. With no column in system, pumps A and B are primed with buffer C.
  2. Pressure limits are set on both pumps to equal values.
  3. Superdex 200 10/300GL is connected, and the system is operated at a flow rate of 0.5 mL/min.
  4. Wash the sample loading loop with buffer C.
  5. 200μL of sample is loaded at approximately 2mg/mL.
  6. Collect eluting proteins and collect at A280. Noting elution volumes of proteins.
3.4 Automated AKTA FPLC
  1. Sample loop is filled partially or completely with sample.
  2. Two separate buffers are connected to A and B pump P-920 modules for mixing. The output flow is directed from the pump to Mixer M-925.

References

Madadlou A, O'Sullivan S, Sheehan D. Fast Protein Liquid Chromatography. Methods Mol Biol. 2017;1485:365-373. doi: 10.1007/978-1-4939-6412-3_19. PMID: 27730563.

Delaplace P, van der Wal F, Dierick JF, Cordewener JH, Fauconnier ML, du Jardin P, America AH. Potato tuber proteomics: comparison of two complementary extraction methods designed for 2-DE of acidic proteins. Proteomics. 2006 Dec;6(24):6494-7. doi: 10.1002/pmic.200600493. PMID: 17096317.

Yevtushenko DP, Misra S. Spatiotemporal activities of Douglas-fir BiP Pro1 promoter in transgenic potato. Planta. 2018 Dec;248(6):1569-1579. doi: 10.1007/s00425-018-3013-8. Epub 2018 Oct 1. Erratum in: Planta. 2018 Oct 19;: PMID: 30276470.