Team:Beijing 4ELEVEN/Contribution
In our AMPs production system in Pichia pastoris GS115, we choose AOX1 promoter (BBa_I764001), which is a common inducible promoter used in yeast cells, to control the expression. To make sure the AOX1 promoter works properly, we tested its performance with sfGFP.
We added sfGFP gene behind AOX1 promoter, inserted the whole sequence into pPIC9K backbone and transformed the plasmid into P. pastoris GS115. The recombinant strain was conducted fermentation test BMMY Medium. We measured the OD600 absorbance and fluorescence of the fermentation broth every 24 hours, and the supernatant samples during the fermentation were verified through SDS-PAGE gel electrophoresis.
The OD600 absorbance of the recombinant strain that contains sfGFP is a little lower than the control strain P. pastoris GS115. That may be caused by the additional expression of sfGFP, the results shows that expression of heterologous gene would repress cell growth, while the repression is not intensive.
Figure 1. OD600 absorbance of P. pastoris GS115 and recombinant strain that contains sfGFP every 24 hours
The fluorescence of recombinant strain that contains sfGFP turns higher for every 24 hours, while that of P. pastoris GS115 remains mostly the same. This shows that AOX1 promoter under the control of methanol does have the capability of promoting the expression of fluorescent protein.
Figure 2. Fluorescence of P. pastoris GS115 and recombinant strain that contains sfGFP every 24 hours.
From the results (Figure 3), correct bands of sfGFP were shown as expected. The protein band at 24 h can be barely seen; 48 h is greater; and 72, 96, 120 h are nearly the same, all greater than 48 h. So the clarity of the bands increases over time, which means the protein expression is getting more intensive every 24 hours. All the results prove that AOX1 promoter works properly.
Figure 3. SDS-PAGE gel analysis of supernatant samples during the fermentation.
