Solubility tests
The indication that the CRY2/CIB1-Cre-recombinase device may not work properly in E. coli. sent us searching for issues that may prevent functionality of the CRY2 and CIB1 proteins. Due to the potential difficulties of expressing eukaryotic genes CRY2 and CIB1 in E. coli, we determined to test for the solubility of CRY2/CIB1 constructs. Insoluble CRY2 or CIB1 proteins would explain the non-functionality of our devices.
We tested for protein solubility with an SDS PAGE analysis of CRY2-CreN and CIB1-CreC fragments constructed during our initial Cre-recombinase activity tests.
Using an E. coli BL21 and empty pET16 plasmid as controls, we checked for the size of the CRY2-CreN and CIB1-CreC proteins using a Coomassie stain. A 70kDa fragment was detected in the Lysed sample (column 3), an equivalent size to that expected for CRY2-CreN. Much of this fragment was insoluble, (only 5% was soluble) suggesting that CRY2-CreN fragments may be misfolded in E. coli expression, thus limiting potential CRY2 activity within the cell. These results reflected the results recorded for lack of activity for our CRY2-CreN/CIB1-CreC device tested in BL21 (DE3) E. coli using an RFP reporter.
After transforming the construct to BL21 E. coli and culturing with IPTG induction, a very low expression level was observed as determined by the fluorescence of the peaks being comparable to background lysate. Excitation at 400 nm produced a small CFP and YFP peak, and blue light induction for 10 min showed no observable change in the spectrum. Unfortunately the individual fusion proteins could not be separated or purified due to the location of the purification tags.
Significantly, CIB1-CreC fragments during the SDS PAGE analysis showed (column 4) 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 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.
Failure to record Cre-recombinase activity in our device and the SDS PAGE analysis suggesting that our devices were insoluble led us to redesign our construct. This included separating the CRY2 and CIB1 into separate plasmids fused to eCFP and eYFP respectively. (See FRET Results for more information)