Difference between revisions of "Team:ANU-Canberra/solubility"

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<h2>Solubility of FRET constructs</h2>
 
<h2>Solubility of FRET constructs</h2>
 
</header>
 
</header>
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<p>We purified CFP-CRY2 and CIB1-YFP from <i>E. coli</i>. YFP expression was immediately obvious - the soluble fraction was highlighter yellow. CRP expression took a bit more effort, though we eventually expressed the construct successfully. However, the soluble fraction was only weakly fluorescent, though the insoluble pellet was strongly fluorescent. This suggested that CFP-CRY2 was insoluble, which may be due to protein misfolding, aggregation or precipitation.</p>
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<p>We then ran an SDS-PAGE gel on the soluble fractions of CFP-CRY2 and the insoluble fraction of CFP-CRY2. For CIB1-YFP, a weak band at the expected size of 47kDa was observed, however much stronger bands at lower molecular weights were observed - this may indicate proteolytic products reflecting cleavage of either CIB1, YFP or intervening regions (e.g. linkers). Expected band of around 80kDa was not observed for the soluble fraction of CFP-CRY2 (which may still have been too dilute despite concentration of the solution) nor the insoluble (as the lane was messy).</p>
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                                    <img src="https://2015.igem.org/File:SDS_Page.png"></img>
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<p><b>SDS-PAGE of FRET constructs.</b> <i>Lane 1: protein markers, 2: CIB1-YFP (soluble fraction), 3: CFP-CRY2 (soluble fraction), 4: CFP-CRY2 (insoluble fraction). Stained with Coomassie Blue.</i></p>                           
 
<header>
 
<header>
 
<h2>Solubility of Cre-recombinase constructs</h2>
 
<h2>Solubility of Cre-recombinase constructs</h2>
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Significantly, CIB1-CreC fragments during the SDS PAGE analysis (column 4) suggested proteolysis of the mainly soluble fragment or a lack of full-length protein expression (as a band at 47kDa was expected).  This was particularly puzzling as our research into the CRY2/CIB1 optogentic device had suggested that CIB1 expressed successfully in <i>E. coli</i>. It was suggested by our Instructor (Spencer Whitney) that the large spans of lysine residues occuring on the CIB1 fragment may have made CIB1 particularly prone to proteolysis.  
 
Significantly, CIB1-CreC fragments during the SDS PAGE analysis (column 4) suggested proteolysis of the mainly soluble fragment or a lack of full-length protein expression (as a band at 47kDa was expected).  This was particularly puzzling as our research into the CRY2/CIB1 optogentic device had suggested that CIB1 expressed successfully in <i>E. coli</i>. It was suggested by our Instructor (Spencer Whitney) that the large spans of lysine residues occuring on the CIB1 fragment may have made CIB1 particularly prone to proteolysis.  
 
                                     </p>
 
                                     </p>
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                                     <img src="https://static.igem.org/mediawiki/2015/6/67/Solubility.png"></img>                          
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                                     <img src="https://static.igem.org/mediawiki/2015/6/67/Solubility.png"></img>
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<p><b>SDS-PAGE (upper) and Western Blot (lower) of Cre-recombinase constructs.</b> <i>Gel stained with Coomassie Blue.</i></p>                             
 
                                     <p>
 
                                     <p>
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<header>
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<h2>Solubility - conclusions</h2>
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</header>
 
Insolubility of CRY2 and proteolysis of CIB1 were consistent across the FRET and Cre-recombinase constructs, suggesting the issue was with those proteins and not a characteristic of the fusion. So although the protein fragments fused to CRY2 and CIB1 may be intact, soluble and functional, it is likely the system would not be light-inducible as the insoluble CRY2 and cleaved CIB1 cannot associate.
 
Insolubility of CRY2 and proteolysis of CIB1 were consistent across the FRET and Cre-recombinase constructs, suggesting the issue was with those proteins and not a characteristic of the fusion. So although the protein fragments fused to CRY2 and CIB1 may be intact, soluble and functional, it is likely the system would not be light-inducible as the insoluble CRY2 and cleaved CIB1 cannot associate.
 
</p>
 
</p>

Revision as of 14:09, 18 September 2015

Solubility of CRY2 and CIB1

Due to the potential difficulties of expressing eukaryotic genes (the A. thaliana-derived CRY2 and CIB1) in prokaryotes, we used SDS-PAGE analyses to test whether the CRY2 and CIB1 fusion proteins were intact and soluble in E. coli. Insolubility of CRY2 or CIB1 could flag to us whether our system could associate under light and function properly.

Solubility of FRET constructs

We purified CFP-CRY2 and CIB1-YFP from E. coli. YFP expression was immediately obvious - the soluble fraction was highlighter yellow. CRP expression took a bit more effort, though we eventually expressed the construct successfully. However, the soluble fraction was only weakly fluorescent, though the insoluble pellet was strongly fluorescent. This suggested that CFP-CRY2 was insoluble, which may be due to protein misfolding, aggregation or precipitation.

We then ran an SDS-PAGE gel on the soluble fractions of CFP-CRY2 and the insoluble fraction of CFP-CRY2. For CIB1-YFP, a weak band at the expected size of 47kDa was observed, however much stronger bands at lower molecular weights were observed - this may indicate proteolytic products reflecting cleavage of either CIB1, YFP or intervening regions (e.g. linkers). Expected band of around 80kDa was not observed for the soluble fraction of CFP-CRY2 (which may still have been too dilute despite concentration of the solution) nor the insoluble (as the lane was messy).

SDS-PAGE of FRET constructs. Lane 1: protein markers, 2: CIB1-YFP (soluble fraction), 3: CFP-CRY2 (soluble fraction), 4: CFP-CRY2 (insoluble fraction). Stained with Coomassie Blue.

Solubility of Cre-recombinase constructs

We checked for the size of the CRY2-CreN and CIB1-CreC proteins using a Coomassie stain (using BL21 cells and empty pET16 vector as controls). A 70kDa fragment was detected in the lysed sample (column 3), the expected size of CRY2-CreN. Much of this fragment was insoluble (only 5% was soluble) suggesting that CRY2-CreN fragments may be misfolded or aggregated, thus limiting potential CRY2 activity within the cell.

Significantly, CIB1-CreC fragments during the SDS PAGE analysis (column 4) suggested proteolysis of the mainly soluble fragment or a lack of full-length protein expression (as a band at 47kDa was expected). This was particularly puzzling as our research into the CRY2/CIB1 optogentic device had suggested that CIB1 expressed successfully in E. coli. It was suggested by our Instructor (Spencer Whitney) that the large spans of lysine residues occuring on the CIB1 fragment may have made CIB1 particularly prone to proteolysis.

SDS-PAGE (upper) and Western Blot (lower) of Cre-recombinase constructs. Gel stained with Coomassie Blue.

Solubility - conclusions

Insolubility of CRY2 and proteolysis of CIB1 were consistent across the FRET and Cre-recombinase constructs, suggesting the issue was with those proteins and not a characteristic of the fusion. So although the protein fragments fused to CRY2 and CIB1 may be intact, soluble and functional, it is likely the system would not be light-inducible as the insoluble CRY2 and cleaved CIB1 cannot associate.