Name: looking outwards: isolation of cyanobacterial released carbohydrate polymers and proteins
Uploaded: 2019-05-27T13:00:00
Description: Watch this Scientific Journal Video about Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins at JoVE.com

This work was financed by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT - Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia/Minist&#233;rio da Ci&#234;ncia, Tecnologia e Ensino Superior in the framework of the project POCI-01-0145-FEDER-028779 and the grant SFRH/BD/99715/2014 (CF).

1. Cyanobacterial released carbohydrate polymer isolation
<ol>
	<li>Polymer isolation and removal of contaminants 
	<ol>
		<li>Cultivate the cyanobacterial strain under standard conditions [e.g., 30 &#176;C under a 12 h light (50 &#956;E m&#8722;2s&#8722;1)/12 h dark regimen, with orbital shaking at 150 rpm]. Measure growth using standard protocols [e.g., optical density at 730 nm (OD730nm), chlorophyll a, dry-weight, etc.], then measure the production of relea...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+secreted+proteins+of+the+cyanobacterium+Synechocystis+sp.+strain+PCC+6803.">Identification of secreted proteins of the cyanobacterium Synechocystis sp. strain PCC 6803.</a> FEMS Microbiology Letters. 193 (2), 213-216 (2000).</li><li>Oliveira, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatile+TolC-like+Slr1270+in+the+cyanobacterium+Synechocystis+sp.+PCC+6803.">The versatile TolC-like Slr1270 in the cyanobacterium Synechocystis sp. PCC 6803.</a> Environmental Microbiology. 18 (2), 486-502 (2016).</li><li>Flores, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+alternative+sigma+factor+SigF+is+a+key+player+in+the+control+of+secretion+mechanisms+in+Synechocystis+sp.+PCC+6803.">The alternative sigma factor SigF is a key player in the control of secretion mechanisms in Synechocystis sp. PCC 6803.</a> Environmental Microbiology. 21 (1), 343-359 (2018).</li><li>Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., Smith, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+method+for+determination+of+sugars+and+related+substances.">Colorimetric method for determination of sugars and related substances.</a> Analytical Chemistry. 28 (3), 350-356 (1956).</li><li>Parikh, A., Madamwar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+characterization+of+extracellular+polysaccharides+from+cyanobacteria.">Partial characterization of extracellular polysaccharides from cyanobacteria.</a> Bioresource Technology. 97 (15), 1822-1827 (2006).</li><li>R&#252;hmann, B., Schmid, J., Sieber, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+identify+the+unexplored+diversity+of+microbial+exopolysaccharides.">Methods to identify the unexplored diversity of microbial exopolysaccharides.</a> Frontiers in Microbiology. 6, 565 (2015).</li><li>Pathak, J., Rajneesh, R., Sonker, A. 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To better understand bacterial secretion mechanisms and study the released products, it is of extreme importance to demonstrate the efficient isolation and analysis of the biomolecules present in the extracellular bacterial environment (such as released carbohydrate polymers and proteins).
Cyanobacterial extracellular carbohydrate polymers are extremely complex, mainly due to the number and proportion of different monosaccharides that constitute their composition1. The ...

Cyanobacteria are widely recognized as prolific sources of natural products with promising biotechnological/biomedical applications. Therefore, understanding cyanobacterial secretion mechanisms and optimization of the extraction/recovery methods are essential to implement cyanobacteria as efficient microbial cell factories.
Many cyanobacterial strains are able to produce extracellular polymeric substances (EPS), mainly formed by heteropolysaccharides, that remain associated to the cell surface or are released into the medium1. These released carbohydrate polymers have distinct features compared to those from other bacteria, which make them suitable for a wide range of applications (e.g., antivirals2, immunostimulatory3, antioxidant4, metal-chelating5, emulsifying6, and drug delivery agents7,8). Methodology for the isolation of these polymers largely contributes&#160;not only to improved yield but also to increased purity and the specific physical properties of the polymer obtained9. A vast majority of these methods for isolation of the polymers rely on precipitation strategies from the culture medium that are easily accomplished due to the polymer&#8217;s strong anionic nature9,10. In addition, removal of the solvents used in the precipitation step can be rapidly achieved by evaporation and/or lyophilization. Depending on the foreseen application, different steps can be coupled either after or before polymer precipitation in order to tailor the final product, which include trichloroacetic acid (TCA) treatment, filtration, or size exclusion chromatography (SEC) column purification10.
Cyanobacteria are also able to secrete a wide range of proteins through pathways dependent on membrane transporters (classical)11 or mediated by vesicles (non-classical)12. Therefore, analysis of the cyanobacterial exoproteome constitutes an essential tool, both to understand/manipulate cyanobacterial protein secretion mechanisms and understand the specific extracellular function of these proteins. Reliable isolation and analysis of exoproteomes require the concentration of the extracellular milieu, since the abundance of secreted proteins is relatively low. In addition, other physical or chemical steps (e.g., centrifugation, filtration, or protein precipitation) may optimize the quality of the exoproteome obtained, enriching the protein content13, and avoiding the presence of contaminants (e.g., pigments, carbohydrates, etc.)14,15 or the predominance of intracellular proteins in the samples. However, some of these steps may also restrict the set of proteins that can be detected, leading to a biased analysis.
This work describes efficient protocols for the isolation of released carbohydrate polymers and exoproteomes from cyanobacteria culture media. These protocols can be easily adapted to the study&#8217;s specific objectives and user needs, while maintaining the basic steps presented here.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Dialysis membranes</td>
			<td style="width:191px;">Medicell Membranes Ltd&#160;</td>
			<td style="width:119px;">DTV.12000.07</td>
			<td style="width:777px;">Visking Tubing Size 7, Dia 23.8 mm, Width 39-41 mm 30m Roll&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Ethanol 96%</td>
			<td style="width:191px;">AGA - &#193;lcool e G&#233;neros Alimentares, S.A.</td>
			<td style="width:119px;">4.000.02.02.00</td>
			<td>Fermentation ethyl alcohol 96% AGA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">PES Filter 0.2 &#956;m</td>
			<td style="width:191px;">Fisher Scientific, Lda</td>
			<td style="width:119px;">15206869</td>
			<td>Syringe filter polystyrene 33MM 0.2&#181;M STR&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Amicon Ultra-15, Ultracel-3K</td>
			<td style="width:191px;">Merck Millipore Ltd.</td>
			<td style="width:119px;">UFC900324</td>
			<td>Centrifugal filters with a nominal molecular weight cut-off of 3 kDa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Thermo Scientific&#160;Pierce BCA Protein Assay</td>
			<td style="width:191px;">Fisher Scientific, Lda</td>
			<td style="width:119px;">10741395</td>
			<td>Green-to-blue, precise, detergent-compatible assay reagent to measure total protein concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brillant Blue G Colloidal Concentrate&#160;</td>
			<td style="width:191px;">Sigma Aldrich Qu&#237;mica SL</td>
			<td style="width:119px;">B2025-1EA</td>
			<td>Coomassie blue&#160;</td>
		</tr>
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looking outwards: isolation of cyanobacterial released carbohydrate polymers and proteins

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The identification and characterization of these biomolecules can improve knowledge about their secretion pathways and help to manipulate them. Furthermore, some of these biomolecules are also interesting in terms of biotechnological applications. Described here are two protocols for easy and rapid isolation of cyanobacterial released carbohydrate polymers and proteins. The method for isolation of released carbohydrate polymers is based on conventional precipitation techniques of polysaccharides in aqueous solutions using organic solvents. This method preserves the characteristics of the polymer and simultaneously avoids the presence of contaminants from cell debris and culture medium. At the end of the process, the lyophilized polymer is ready to be used or characterized or can be subjected to further rounds of purification, depending on the final intended use. Regarding the isolation of the cyanobacterial exoproteome, the technique is based on the concentration of the cell-free medium after removal of the major contaminants by centrifugation and filtration. This strategy allows for reliable isolation of proteins that reach the extracellular milieu via membrane transporters or outer membrane vesicles. These proteins can be subsequently identified using standard mass spectrometry techniques. The protocols presented here can be applied not only to a wide range of cyanobacteria, but also to other bacterial strains. Furthermore, these procedures can be easily tailored according to the final use of the products, purity degree required, and bacterial strain.

A schematic representation of the method described to extract released carbohydrate polymers from cyanobacterial cultures is depicted in Figure 1. Precipitated polymers from the moderate EPS producer cyanobacterium Synechocystis sp. PCC 6803 and the efficient EPS producer Cyanothece sp. CCY 0110 are shown in Figure 2. In Figure 3, lyophilized polymers with different degrees of contamination are shown, highlighting ...

Watch this Scientific Journal Video about Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins at JoVE.com

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

Here, protocols for the isolation of cyanobacterial released carbohydrate polymers and isolation of their exoproteomes are described. Both procedures embody key steps to obtain polymers or proteins with high purity degrees that can be used for further analysis or applications. They can also be easily adapted according to specific user needs.

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The ...

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