Team:NJTech China/Contribution

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Contribution

While most of our project was focused on Design and Construction of Synthetic Yeast-Microalgae Consortia for Biosynthesis of Phenylethanol, we were also interested in chromoproteins. Specifically, we characterized the expression of aeBlue and gfasPurple.

The aeBlue sequence (Part:BBa_K864401) optimized for E. coli was incorporated into plasmid pET-29a(+), transformed into E. coli BL21 for characterization and measurement. We provided aeBlue with results and data based on protein expression and purification, TOF-Mass spectrometry, full wavelength measurement and Swiss-model.

Methods

SDS-PAGE, TOF-Mass Spectrometry, BCA (Bicinchoninic acid) method, full wavelength measurement and Swiss-Model.

Results

Fig. 1 The fermentation broth of aeBlue

Fig. 2 The cell pellet collected after centrifugation

Conclusion

The cell pellet was collected by harvesting 50mL culture after 24h of induction followed by centrifugation at 4 degrees and 6000 rpm for 10min. Then, we performed ultrasonic disruption and collected the supernatant after centrifugation. The protein was purified and collected through ultrafiltration and affinity chromatography.

Conclusion

The protein gel preliminarily proved that the molecular mass of the aeBlue protein was correct, which is consistent with the expected molecular mass of aeBlue protein (the molecular mass of aeBlue protein is about 27.3 kDa). Compared with lane 1, 2 and 3, lane 4, 5, and 6 indicate that more aeBlue protein can be obtained with IPTG induction. As is shown in lane 7, the concentration of protein was increased after ultrafiltration concentration. Lane 8 shows that the purification effect of protein after nickel affinity chromatography was better, and the impurity protein was less than before affinity chromatography. In conclusion, it can be seen that our expression and purification strategy is effective.

Fig. 3 SDS-PAGE of the chromoprotein aeBlue

1. aeBlue- The culture without IPTG induction.

2. aeBlue- Supernatant without IPTG induction after sonication.

3. aeBlue- The sedimentation without IPTG induction and after sonication.

4. aeBlue- Supernatant sample after IPTG induction and sonication.

5. aeBlue- The sedimentation after IPTG induction and ultrasound.

6. aeBlue- The culture after IPTG induction.

7. aeBlue- Protein sample after the ultrafiltration (diluted 5 times).

8. aeBlue- Protein after GST affinity chromatography.

9. aeBlue- Purified protein sample.

We used the BCA (Bicinchoninic acid) method to measure the concentration of aeBlue protein. The concentration of aeblue chromoprotein is 9.10 mg/ml.

It comes out that: The concentration of aeBlue is 9.10 mg/ml.

Fig. 4 BCA Protein Standard Curve

Conclusion

We performed Time of Flight Mass Spectrometer on the purified HIS-tagged aeBlue protein. The predicted molecular mass of this protein is about 27300Da. The result of TOF-Mass Spectrometry showed that the specific molecular mass of aeBlue protein is 27.279kDa (the value of the sharpest peak is shown as the molecular mass of aeBlue protein). Moreover, the intensity of 27.279kDa is up to 1.5×10⁵, which indicates the high concentration and purity of the aeBlue protein. There are also some small protein peaks, suggesting that the noise had some effect, but not much.

Fig. 5 TOF MS of aeBlue

aeBlue protein full-wavelength scan profile :

1-204nm 0.282A         3-272nm 0.322A

2-598nm 2.446A         4-1040nm 0.003A

Conclusion

The full-wavelength scan of aeBlue protein shows that the strongest absorption peak of aeBlue protein occurs at 598nm. As shown in the results, aeBlue has a low intensity peak at 204 to 272 nm, which may be due to the fluorescence excitation.

Fig. 6 Absorption spectrum of aeBlue (190-1100nm)

Structural modeling results of the aeBlue protein based on Swiss-Model

Fig. 7 The results of the homology and structural modelling protein aeBlue

Conclusion

We used Swiss-Model to simulate the three-dimensional structure of aeBlue protein. The above figures showed the modeling result of Swiss-Model.

The gfasPurple sequence (Part:BBa_K1033918) optimized for E. coli was incorporated into E. coli BL21 for protein characterization and data measurement. We conducted a series of experiments to obtain new data of gfasPurple chromoprotein.

Methods

We performed SDS-PAGE on the gfasPurple protein to screen the protein expression and detect the effect of purification. Next, we performed the Time of Flight Mass Spectrometer for the gfasPurple protein sample. We also applied the full wavelength measurement on the gfasPurple protein. Finally, we used Swiss-Model to simulate the three-dimensional structure of the protein.

Results

Fig. 1 The fermentation broth of gfasPurple

Fig. 2 The cell pellet collected after centrifugation

Conclusion

The cell pellet was collected by harvesting 50mL culture after 24h of induction followed by centrifugation at 6000 rpm for 10 min. Then, we performed ultrasonic disruption and collected the supernatant after centrifugation. The protein was purified and collected through ultrafiltration and affinity chromatography.

Conclusion

The protein gel preliminarily proved that the molecular mass of the gfasPurple protein was correct, which is consistent with the expected molecular mass of gfasPurple protein (the molecular mass of gfasPurple protein is about 26.5 kDa). Compared with lane 5, 6, and 7, lanes 1, 2, 3 and 4 indicate that more gfasPurple protein can be obtained with IPTG induction. As is shown in lane 8, the concentration of protein was increased after ultrafiltration concentration. Lane 9 shows that the purification effect of protein after nickel affinity chromatography was better, and the impurity protein was less than before affinity chromatography. In conclusion, it can be seen that our expression and purification strategy is effective.

Fig. 3 SDS-PAGE of the chromoprotein gfasPurple

1. gfasPurple- The culture without IPTG induction.

2. gfasPurple- Supernatant without IPTG induction after sonication.

3. gfasPurple- The sedimentation without IPTG induction and after sonication.

4. gfasPurple- Supernatant sample after IPTG induction and sonication.

5. gfasPurple- The sedimentation after IPTG induction and ultrasound.

6. gfasPurple- The culture after IPTG induction.

7. gfasPurple- Protein sample after the ultrafiltration (diluted 5 times).

8. gfasPurple- Protein after GST affinity chromatography.

9. gfasPurple- Purified protein sample.

Conclusion

We used the BCA (Bicinchoninic acid) method to measure the concentration of gfasPurple protein. The concentration of gfasPurple chromoprotein is 0.2494 mg/ml.

It comes out that: The concentration of gfasPurple is 0.2494 mg/ml.

Fig. 4 BCA Protein Standard Curve

Conclusion

We performed a Time of Flight Mass Spectrometer on the purified HIS-tagged gfasPurple protein. The predicted molecular mass of this protein is about 24.8KDa. The result of TOF-Mass Spectrometry showed that the specific molecular mass of gfasPurple protein is 24.793kDa (the value of the sharpest peak is shown as the molecular mass of gfasPurple protein). Moreover, The highest peak was at 18.246KDa, probably because the protein was degraded during transportation without strict fourth degree insulation.

Fig. 5 TOF MS of gfasPurple

1-286nm 0.449A            3-580nm 0.575A

2-1044nm 0.010A          4-1098nm 0.021A

Conclusion

The full-wavelength scan of gfasPurple protein shows that the strongest absorption. peak of gfasPurple protein occurs at 580nm.

Fig. 6 Absorption spectrum of gfasPurple (190 to 1100nm)

Structural modeling results of the gfasPurple protein based on Swiss-Model

Fig. 7 The results of the homology and structural modelling protein gfasPurple

Conclusion

We used Swiss-Model to simulate the three-dimensional structure of gfasPurple protein. The above figures showed the modeling result of Swiss-Model.