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 total cell and soluble fractions of CFP-CRY2 and CIB1-YFP expressing cells. 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 most likely of CIB1 (as strong YFP expression would not be seen if YFP were cleaved). The expected band at 86kDa was observed for CRY2-YFP, as well as a number of truncated products, however the bands in the soluble fraction were much weaker than in the total cell extract, indicating that CRY2-YFP is only slightly soluble in E. coli.
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
