Team:Korea U Seoul/Project/Result

The overall reaction for precipitation is

Ca2++HCO3-→ CaCO3+H+

According to that equation, CaCO3 generated the more the pH will be lowered.
NA, R and CA is cloning different proteins. NA is expressed from full gene of nacrein, R from nacrein's repeated domain and CA from carbonic anhydrase. The effect of NA and R on the rate of precipitation of CaCO3 was determined by recording the decrease in pH of a 20mM Tris HCl pH8.0 buffer solution at 10°C



In this graph shows drop of pH and according to above, drop of pH means CaCO3 is madden. By the result we can knew that there is no differ between 3 samples. Our hypothesis was repeat domain binding with Ca so precipitation will decrease.


However in our data there's no gap between 3 sample. So we make another hypothesis that R or NA is precipitate with CaCO3. Our team start to check that and it will be our further study.



we also check CA activity to check NA and CA can role in carbonic anhydric reaction

By the result we can see that CA activity of NA is biger than formal condition(Tris) but CA is lower. It means NA act as a catalyst but CA act as a inhibitor. It can be explain by le chatelier's principle. Because of transition of carbonic acid caused by CA, reaction equilibrium in carbonic acid fixation by calcium ion is shift from forward to inverse. That's why CaCO3 decrease when CA is treated. But in the case of NA, R domain capture CO3 immediately so carbonic acid is decrease. So reaction equilibrium shift to forward and can be active continue.

We find that nacrein really have a role in fixation of carbon dioxide. Next we want to know how well nacrein express in E coli. We use two E coli, BW25113, BL21(DE3). They are specialized to make protein. Next diagram is explain our experiment.



By the result, sample made by BW25113 and BL21(DE3) can't identify nacrein and repeat domain clearly. We think it caused by insoluble characteristic of made nacrein. So we use pB4 system which can make protein soluble.



We check expression in two condition(37℃ and 16℃). Process shown in next diagram.



we can see identify all proteins by this test. In two condition we can find nacrein, repeat, CA and deletedR. But some proteins can find easier in pellet but others in supernatant. You can check clearly in next picture.

1. nacrein can be act as a catalyst for carbonic anhydrase.
2. nacrein and its composition can made in vitro.
3. we can make nacrein both soluble and insoluble form.