Part:BBa_K1582025
Thc_Cut1+sJanus-m Fusion Protein
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1048
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1057
Protein Expression
Protein pre-expression experimental conditions:
1.Transferred our correct plasmid into escherichia coli BL21 (DE3).
2.Select bacterial colony and add into LB, incubate at 37 centigrade for 7h. Add 4μl of IPTG to induce the expression of protein.
3.Incubate at 37 centigrade for 4h. The bacterial solution’s OD is 0.6-0.8.
Figure 1. The result of protein Thc_Cut1-sJanus-m pre-expression. Labels are the same as before.
Finally, the protein was expressed successfully.
We added 5μl of the bacterial restored into the LB containing 5μl of ampicillin to the final concentration of 1mM. And then, we incubated them in the shaker at the temperature of 37 centigrade, working for 14-16 hours.
Experimental conditions as follow：
1. Incubate at 37 centigrade, until OD ranges from 0.6-0.8(4-5h).
2. Incubate at 4 centigrade, 220rpm, for 30mins.
3. Add 1mL IPTG to the final concentration of 1mM.
4. Incubate at 16 centigrade for 12-16h.
The purification of protein:
We used eppendorf to make bacterial deposited, working at 4000rpm for 20min. Use 15mL MCAC0 to suspend bacterial.
After resuspending, we used high pressure to break the cells.
In order to get the recombinant protein with the higher purity, the recombinant protein was purified through Ni-chelating affinity chromatography.
The results are as follow:
Figure 1. The result of protein Thc_Cut1-sJanus-m expression. 1: Sample non-induced; 2: Sample induced; 3: Sample of cytoplasm deposited; 4: Sample which outflowed from Ni column combined with supernatant; 5: Sample of NIi medium combined with supernatant; 6: Sample washed by MCAC20; 7: Sample resuspended by MCAC30; 8: Sample washed by MCAC30; 9: Sample resuspended by MCAC50; 10: Sample washed by MCAC50; 11: Sample resuspended by MCAC100; 12: Sample washed by MCAC100; 13: Sample resuspended by MCAC200; 14: Sample washed by MCAC200; 15: Sample resuspended by MCAC500; 16: Sample washed by MCAC500; 17: Sample resuspended by MCAC1000
Stimulated Plastic Ezymolysis
Further exploration
Overview
We successfully build a fusion protein which attach cutinase Thc_Cut1 to our Janus (sJanus-m). The fusion protein has much more better performance than the cutinase before.
Result
The procedure in this part is likely to the pre-experiment, we use different concentration of fusion protein. And we compare the data to the value we conduct with Thc_Cut1 before, found that fusion protein hydrolysis more, especially in high concentration.
Figure 1. These curves compare fusion protein’s performance with normal cutinase in different concentration. We can see that fusion protein perform much better when concentration are over 1mg/ml.
We also conduct our experiments in different pH by using different Tris-HCl. Just as picture shows, the activity of our fusion protein doesn’t change a lot in different pH which is similar with Thc_Cut1.
Figure 1. These two proteins are stable when pH changes, especially when pH is between 6.0 and 9.0. And the concentration of Thc_Cut1 and Thc_Cut1-sJanus-m are 0.2mg/ml.
We drew a curve about detect time and compared it with Thc_Cut1, we can see the hydrolysis rate rose sharply after 5h,while Thc_Cut1 still in the low level. Which represent our Janus works well!
And our fusion protein does improve a lot!
Figure 1. We detect absorbance of liquid every 3h, red line refers to our fusion protein, and the blue line is former enzyme. The performance of cutinase are highly improved when fuse to the sJanus-m.
Figure 1. The picture shows 5mg/ml enzyme have react with one piece of plastic（3*3*0.25mm）for 3h, before and after centrifugation. We can see clearly in the pic. After 3hs the tube of Thc_Cut1-sJanus-m had much more sediment than Thc_Cut1.
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