Jun 29, 2020

Public workspaceIon-exchange purification of fucoidans

This protocol is a draft, published without a DOI.
  • 1(Marum of the University of Bremen)
Andreas Sichert
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Protocol Citation: Andreas Sichert, Jan-Hendrik Hehemann 2020. Ion-exchange purification of fucoidans. protocols.io https://protocols.io/view/ion-exchange-purification-of-fucoidans-8axhsfn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 16, 2019
Last Modified: June 29, 2020
Protocol Integer ID: 28727
Keywords: Fucoidan, Ion-exchange chromatography, Brown algae, Sulfated polysaccharides,
Abstract
Fucoidans are a diverse class of sulfated polysaccharides integral to the cell wall of brown algae and due to their various bioactivities, they are potential drugs. Standardized work with fucoidans is required for structure-function studies, but remains challenging since available fucoidan preparations are often contaminated with other algal compounds. Additionally, fucoidans are structurally diverse depending on species and season, urging the need for standardized purification protocols. Here, we use ion-exchange chromatography to purify different fucoidans and found a high structural diversity between fucoidans. Ion-exchange chromatography efficiently removes the polysaccharides alginate and laminarin and other contaminants such as proteins and phlorotannins across a broad range of fucoidans from major brown algal orders including Ectocarpales, Laminarinales and Fucales. By monomer composition, linkage analysis and NMR characterization, we identified glucuronic acid and O-acetylation as new structural features of certain fucoidans and provide a novel structure of fucoidan from Durvillaea potatorum with a-1,3 linked fucose backbone and b-1,6 and b-1,3 galactose branches. This study emphasizes the use of standardized ion-exchange chromatography to obtain defined fucoidans for subsequent molecular studies.
Materials
MATERIALS
ReagentSodium ChlorideCatalog #PubChem CID: 5234
ReagentTris (Tris Base)Gold BiotechnologyCatalog #T-400
ReagentNalgene™ Rapid-Flow™ Sterile Disposable Bottle Top Filters with PES Membrane, 1000mL, 0.2μm pore, 33mm neckThermo FisherCatalog #597-3320
ReagentANX Sepharose 4 Fast FlowGE HealthcareCatalog #17128701
ReagentXK 50/20 ColumnGE HealthcareCatalog #28988952
ReagentFucus serratus fucoidanCarbosynthCatalog #YF157167
ReagentEcklonia maxima fucoidanCarbosynthCatalog #YF157166
ReagentSargassum fusiforme fucoidanCarbosynthCatalog #YF157167
ReagentMacrocystis pyrifera fucoidanCarbosynthCatalog #YF145109
ReagentCladosiphon okamuranus fucoidanCarbosynthCatalog #YF146834
ReagentDurvillaea potatorum fucoidanCarbosynthCatalog #YF157165
ReagentLessonia nigrescens fucoidanCarbosynthCatalog #YF146833
Simple chromatography setup
Simple chromatography setup
1h
1h
Prepare a simple chromatography system consisting of:
  1. MasterFlex L/S peristaltic pump
  2. XK50/250 column (GE healthcare)
  3. Suitable tubings and connectors
  4. Slurry 200 mL of the ANX and pack it into the column
  5. Compress the resin by applying a gentle flow (~5 mL/min)

Download IEX_setup.pngIEX_setup.png

Buffer preparation
Buffer preparation
1h
1h
  1. ~2 liters of double de-ionized water (ddH2O)
  2. ~2 liters of 50 mM Tris-HCl pH=7.5 in ddH2O (Buffer A)
  3. ~1 liters of 50 mM Tris-HCl pH=7.5 in ddH2O with 0.5 M NaCl (Buffer B)
  4. ~1 liters of 50 mM Tris-HCl pH=7.5 in ddH2O with 5 M NaCl (Buffer C)
  5. ~1 liters of250 mM NaOH in ddH2O
Filter all buffers through a 0.2 μm bottle top filter to remove dust
Rinse filters with ddH2O and dry for reuse
Prepare polysaccharide solution
Prepare polysaccharide solution
4h
4h
Mix 0.5 g fucoidan in 500 mL of 50 mM Tris pH 7.5 in ddH2O
Use a magnetic stirrer and heat ~50-60°C for ~1h or until fucoidans dissolved
Transfer solution into centrifuge vials and spin down at Centrifigation4500 x g , for 30'

Carefully decant supernatant into new bottle, avoid transfering solids

Chromatography
Chromatography
20m
20m
Set the flow of the pump to 40-50 mL/min
Equilibrate the column with 3 CV Buffer A
Apply fucoidan in a semi-batch mode. For this, connect the outlet tube to your input reservoir to circulate the solution at least 3x over the column.
Wash with 1 CV Buffer A
Wash with 3 CV Buffer B
Elute with 0.5 CV Buffer C
Keep in mind the void volumn of your column (~200 mL). After switching to Buffer C, you need to wait one CV. The high salt concetration of Buffer C results in visible schlieren in a low salt buffer.
Critical
Collect the first 100 mL of eluted Buffer C
Proceed with the eluted fraction to the dialysis

Wash column
2 CV Buffer C
2 CV ddH2O
2 CV NaOH
2 CV ddH2O or until pH is neutral
For long-term storage of the column, use 20% Ethanol in ddH20
Post processing
Post processing
2h
2h
Transfer the eluted polysaccharide fraction into a 1 kDa dialysis tubing
Dialyse against ddH2O and change the ddH2O several times
Transfer the liquid into a new vial
Freeze-dry the sample