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While most of our project was focused on signaling pathways of yeasts, we were also interested in chromoproteins. Specifically, we characterized the expression of amajLime and eforRed.

The amajLime sequence (Part:BBa_K1033916) optimized for E. coli was incorporated into plasmid pET-28a(+), transformed into E. coli BL21 for characterization and measurement. We provided amajLime 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 amajLime

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.

Fig. 3 SDS-PAGE of the chromoprotein amajLime

1. amajLime- The culture after IPTG induction.

2. amajLime- The pellet after IPTG induction and ultrasound.

3. amajLime- Supernatant after IPTG induction and sonication.

4. amajLime- The culture without IPTG induction.

5. amajLime- The pellet without IPTG induction after ultrasound.

6. amajLime- Supernatant sample without IPTG induction after sonication.

7. amajLime- Protein sample after the ultrafiltration (diluted 20 times).

8. amajLime- Purified protein sample.

Conclusion

The protein gel preliminarily proved that the molecular mass of the amajLime protein was correct, which is consistent with the expected molecular mass of amajLime protein (the molecular mass of amajLime protein is about 26.8 kDa). Compared with lane 4, 5 and 6, lane 1, 2, and 3 indicate that more amajLime 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. 4 BCA Protein Standard Curve

We used the BCA (Bicinchoninic acid) method to measure the concentration of amajLime protein. The concentration of blue chromoprotein was 0.6608 mg/ml.

It comes out that: The concentration of amajLime is 0.6608 mg/ml

Fig. 5 TOF MS of amajLime

Conclusion

We performed Time of Flight Mass Spectrometer on the purified HIS-tagged amajLime protein. The predicted molecular mass of this protein is about 26840Da. The result of TOF-Mass Spectrometry showed that the specific molecular mass of amajLime protein is 26.898kDa (the value of the sharpest peak is shown as the molecular mass of amajLime protein). Moreover, the intensity of 26.898kDa is up to 4x105, which indicates the high concentration and purity of the amajLime protein. There are also some small protein peaks, suggesting that the noise had some effect, but not much.

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

amajLime protein full-wavelength scan profile :

1-198nm 2.551A          3-276nm 0.146A

2-210nm 2.683A          4-454nm 0.135A

Conclusion

The full-wavelength scan of amajLime protein shows that the strongest absorption peak of amajLime protein occurs at 210nm. As shown in the results, amajLime has a low intensity peak at 400 to 450 nm, which may be due to the fluorescence excitation demonstrated by previous teams such as Hong Kong-CUHK iGEM 2017.

Fig. 7 Absorption spectrum of amajLime (350-660nm)

Conclusion

The full wavelength measurement of amajLime (359-660nm) was compared with the excitation spectrum figure of 2013UPPSALA , indicating that the results of amajLime protein characterized by our team were similar to those of 2013 UPPSALA and Hong Kong-CUHK iGEM 2017.

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

Fig. 8 The results of the homology and structural modelling protein amajLime

Conclusion

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

The eforRed sequence (Part:BBa_K592012) 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 eforRed chromoprotein.

Methods

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

Results

Fig. 1 The fermentation broth of eforRed

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.

Fig. 3 SDS-PAGE of the chromoprotein eforRed

1. eforRed- The culture after IPTG induction.

2. eforRed- The culture without IPTG induction.

3. eforRed- Supernatant after IPTG induction and sonication.

4. eforRed- Supernatant sample without IPTG induction after sonication.

5. eforRed- The pellet after IPTG induction and ultrasound.

6. eforRed- The pellet without IPTG induction after ultrasound.

7. eforRed- Protein sample before affinity chromatography.

8. eforRed- Purified protein sample.

9. eforRed- Protein sample after concentration.

Conclusion

The protein gel proved that the molecular mass of the protein was correct (the molecular mass of eforRed protein is about 27kDa), and more eforRed protein can be obtained with IPTG induction (compare lanes 1, 3, 5 with lanes 2, 4, and 6). The protein purification after nickel affinity chromatography was effective (lane 8). The impurity protein was less than the sample before affinity chromatography (lanes 7, 8).

Fig. 4 TOF MS of eforRed

Conclusion

We performed TOF-Mass Spectrometry on the purified eforRed chromoprotein. The predicted molecular mass of this protein is about 27kDa, and the exact size is 27306.3Da (the value of the highest peak is shown as the molecular weight of eforRed protein). The other lines are impurity proteins, which may because the nickel affinity chromatography did not remove all impurity proteins or the eforRed protein itself degraded into small fragments.

Fig. 5 Absorption spectrum of eforRed (190 to 1100nm)

Conclusion

eforRed protein full-wavelength scan profile :

198nm:2.625A      210nm:2.775A      216nm:2.780A      224nm:2.812A

278nm:0.283A      344nm:0.088A      582nm:0.630A      1066nm:0.017A

The above figure showed the absorption spectrum of eforRed (190 to 1100nm). The full-wavelength scan of eforRed protein showed that the strongest absorption peak of eforRed protein occurs at 224nm.

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

Fig. 6 The results of the homology and structural modelling protein eforRed

Conclusion

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