Figure 1.2 shows the qualitative fluorescence results of our constructs with unmodified 0.19 tagged with GFP and a secretion tag. Figure 1.1 shows our modified 0.19 constructs, also tagged with GFP and a secretion tag. A seen in the photographs above, our modified 0.19 constructs are fluorescent, showing that our modificaitons have not prevented the secretion tag from functioning, thus enabling the modified 0.19 to be secreted from yeast cells.
Confirming the Molecular Weight of 0.19
Figure 2
This is the electrophoresis with 0.19 protein solution(Figure 2).The protein marker in lane 1 and 5 (counting from left) show bands of molecular weight at 10, 13, 22, 32, 67, and 94 kDa. In lane 2 and 3 we have GFP-tagged 0.19 with combined molecular weight of around 40kDa and the electrophoresis results show that the two bands are between 32 and 67 kDa as expected. To confirm the molecular weight of 0.19 by itself, we put the purified protein solution collected from the cell pellet containing Construct 1 in lane 6 and 7. Although the bands are faint, the results show the correct molecular weight of 0.19, which is 13kDa. We assumed that the faint bands are due to the low protein concentration.
2-chloro-4-nitrophenyl maltotrioside (CNP) Assay
Figure 1
Figure #
As you can see from the data above, the α-amylase activity for all of the 0.19 variations α-amylase activity over a 30-minute period was measured. Regarding the modifications made to improve 0.19’s bonding strength to α-amylase (Gly to Glu and Ile to Asn modifications), their α-amylase activity was consistently lower than the original 0.19 throughout the 30-minute period which means the modification that increased the affinity with α-amylase was successful.
The modification with the intention of preventing dimerization, Arg to Glu with and without secretion tag had an α-amylase activity was similar to the unmodified 0.19 from t~3.5 to t=5 throughout the 30-minute period; however, it had a lower α-amylase activity overall, so we considered the modification to be positive. Regarding the modification with the same intention, but for Cys to Arg, there was a higher α-amylase activity than the original 0.19 from t=4 to t=8, but we also consider this to be insignificant according to the overall data, therefore, this modification had a positive result as well.
Looking into the results between the modifications with high affinity to α-amylase, as expected from our results from the dry lab, the Gly to Glu modifications were more effective than the Ile to Asn modifications regarding our modification for strengthening the affinity between 0.19 and α-amylase (solid lines); this can be inferred by comparing the α-amylase activity at t=2 (Gly to Glu without secretion tag was 0.005137 mol/L/min, Gly to Glu with secretion tag was 0.004840 mol/L/min, Ile to Asn without secretion tag was 0.007767 mol/L/min, and Ile to Asn with secretion tag was 0.01354 mol/L/min).
Another point to be mentioned is the increasing slopes in the Arg to Glu modifications from t=2 to t=6 which was different from all the other 0.19s which had a decreasing slope. We were not able to interpret this specific characteristic, so further research is necessary.
The last point that should be noted is the average speed of α-amylase activity in Gly to Glu modification with the secretion tag. Despite the initial α-amylase activity being relatively low compared to the original 0.19 with secretion tag, the average speed of α-amylase activity for Gly to Glu with secretion tag was nearly identical to the average speed of α-amylase activity in the original 0.19 with secretion tag from t=0 to t=2 (Gly to Glu with secretion tag was −0.01499 mol/Lmin, 0.19 with secretion tag was −0.02610 mol/Lmin). This is significant data because the average speed of α-amylase activity is another important factor to consider, and it was interesting to see that only the Gly to Glu with secretion tag had a relatively large average speed of α-amylase activity considering it to start at a relatively low α-amylase activity to the original 0.19s. We do not know the reason behind this, so future investigation is necessary. In conclusion, all of our modifications were overall more successful at inhibiting α-amylase compared to the original 0.19s.
