Skip to main content

The use of immobilised metal affinity chromatography (IMAC) to compare expression of copper-binding proteins in control and copper-exposed marine microalgae

Journal Article


Download full-text (Open Access)

Abstract


  • Toxicity of metals to aquatic organisms is dependent on both external factors, such as exposure concentration and water quality parameters, and intracellular processes including specific metal-binding sites and detoxification. Current models used to predict copper toxicity in microalgae do not adequately consider these intracellular processes. This study compared the copper-binding proteins from four species of marine microalgae, Dunaliella tertiolecta, Tetraselmis sp., Phaedactylum tricornutum and Ceratoneis closterium, in controls (no added copper) and following a 72-h exposure to copper (sufficient to inhibit growth by approximately 50 %). Cells were lysed by sonication, which was optimised to obtain 54–94 % cell rupture for the different algae. Cell lysates were processed by immobilised metal affinity chromatography (IMAC) using Cu2+ as the bound metal (i.e. Cu-IMAC). Bound proteins were subsequently analysed by SDS-PAGE, comparing proteins recovered from algae that were exposed to copper versus untreated control cells. Individual proteins for which copper exposure resulted in changes to proteins present were excised from gels and further analysed by nano LC ESI-MS/MS; proteins were identified using the Mascot database. Proteins identified in this way included heat-shock proteins, rubisco, α- and β-tubulins and ATP synthase (β subunit). The results established that Cu-IMAC is a useful approach to identify proteins involved in copper binding in algae. This study identified several proteins that may play an active role in responses to copper toxicity in marine microalgae.

Publication Date


  • 2014

Citation


  • Smith, C. L., Stauber, J. L., Wilson, M. R. & Jolley, D. F. (2014). The use of immobilised metal affinity chromatography (IMAC) to compare expression of copper-binding proteins in control and copper-exposed marine microalgae. Analytical and Bioanalytical Chemistry, 406 (1), 305-315.

Scopus Eid


  • 2-s2.0-84892952020

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=2432&context=smhpapers

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/1414

Has Global Citation Frequency


Number Of Pages


  • 10

Start Page


  • 305

End Page


  • 315

Volume


  • 406

Issue


  • 1

Place Of Publication


  • Germany

Abstract


  • Toxicity of metals to aquatic organisms is dependent on both external factors, such as exposure concentration and water quality parameters, and intracellular processes including specific metal-binding sites and detoxification. Current models used to predict copper toxicity in microalgae do not adequately consider these intracellular processes. This study compared the copper-binding proteins from four species of marine microalgae, Dunaliella tertiolecta, Tetraselmis sp., Phaedactylum tricornutum and Ceratoneis closterium, in controls (no added copper) and following a 72-h exposure to copper (sufficient to inhibit growth by approximately 50 %). Cells were lysed by sonication, which was optimised to obtain 54–94 % cell rupture for the different algae. Cell lysates were processed by immobilised metal affinity chromatography (IMAC) using Cu2+ as the bound metal (i.e. Cu-IMAC). Bound proteins were subsequently analysed by SDS-PAGE, comparing proteins recovered from algae that were exposed to copper versus untreated control cells. Individual proteins for which copper exposure resulted in changes to proteins present were excised from gels and further analysed by nano LC ESI-MS/MS; proteins were identified using the Mascot database. Proteins identified in this way included heat-shock proteins, rubisco, α- and β-tubulins and ATP synthase (β subunit). The results established that Cu-IMAC is a useful approach to identify proteins involved in copper binding in algae. This study identified several proteins that may play an active role in responses to copper toxicity in marine microalgae.

Publication Date


  • 2014

Citation


  • Smith, C. L., Stauber, J. L., Wilson, M. R. & Jolley, D. F. (2014). The use of immobilised metal affinity chromatography (IMAC) to compare expression of copper-binding proteins in control and copper-exposed marine microalgae. Analytical and Bioanalytical Chemistry, 406 (1), 305-315.

Scopus Eid


  • 2-s2.0-84892952020

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=2432&context=smhpapers

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/1414

Has Global Citation Frequency


Number Of Pages


  • 10

Start Page


  • 305

End Page


  • 315

Volume


  • 406

Issue


  • 1

Place Of Publication


  • Germany