Difference between revisions of "Part:BBa K1582024"

Revision as of 00:52, 24 September 2015

Thc_Cut1+sJanus Fusion Protein

Sequence and Features

Usage

This fusion protein is designed to degrade PET into terephthalic acid and ethylene glycol more powerfully. Compared to single Thc_Cut1, the rate of PET emzymolysis is supposed to be improved by Thc_Cut1-Janus fusion.

Biology

Thc_Cut1 is a kind of cutinase, the details of which can be seen at BBa_K1582028. Class II hydrophobins sJanus are small secreted fungal proteins which can be found in filamentous fungi, which play a role in a broad range of processes in the growth and development of filamentous fungi. Their assembly shows thread-like structure. They could be expressed in prokaryotic cells like E.coli.
According to some research, when Janus is added to the reaction of cutinase and plastics, the reaction rate is supposed to have an obvious improvement.
Based on the above background, we design to use the method of fusion protein to combine cutinase and Janus compactly, through the establishment of fusion protein, we can found if we can further promote the stimulation rate of the plastics degradation. Meanwhile, from the data about fusion protein, we can try to uncover the functional mechanism of the stimulation to the plastics degradation which is unknown until now.

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 expression. 1: Sample non-induced; 2: Sample induced; 3: Sample of cytoplasm deposited; 4: Sample of supernatant; 5: Sample of NIi medium combined with supernatant; 6: Sample which outflowed from Ni column combined with supernatant; 7: Sample washed by MCAC20; 8: Sample resuspended by MCAC30; 9: Sample washed by MCAC30; 10: Sample resuspended by MCAC50; 11: Sample washed by MCAC50; 12: Sample resuspended by MCAC100

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 2. The result of protein Thc_Cut1-sJanus expression. 1: Sample non-induced; 2: Sample induced; 3: Sample of cytoplasm deposited; 4: Sample of supernatant; 5: Sample of NIi medium combined with supernatant; 6: Sample which outflowed from Ni column combined with supernatant; 7: Sample washed by MCAC20; 8: Sample resuspended by MCAC30; 9: Sample washed by MCAC30; 10: Sample resuspended by MCAC50; 11: Sample washed by MCAC50; 12: Sample resuspended by MCAC100

Figure 3. The result of protein Thc_Cut1-sJanus expression.13: Sample washed by MCAC100; 14: Sample resuspended by MCAC200; 15: Sample washed by MCAC200; 16: Sample resuspended by MCAC500; 17: Sample washed by MCAC500; 18: Sample resuspended by MCAC1000.

Protein Extraction Kit

We did the fifth group of experiment. In this experiment, we used Cut1-sJanus to test this system. The concentration of original protein solution is 50ng/mL. The conditions doing this experiment is same as the second group.
Reagents used in this experiment

               Concentration(final)

               Volume

               Berol 532

               Purity 96%

               Protein solution

               50ng/mL

               5mL

Results of this experiment

Figure 4 We used Bandscan5.0 to analysis the gel. We can see clearly that the protein was purified (3 and 6, 4 and 7). Other proteins were removed totally and we get the pure target protein. In this experiment, we used protein having been purified by Nickle chromatography, so the purity of our target protein is high before separating.