I have cloned a fusion protein cassette with a C terminal GFP tag in E.coli based recombinant protein expression system. The expression of the cloned cassette was confirmed by simple visual inspection of the bacterial cell under exposure to blue (UV) light (figure A). The same GFP fusion protein cassette was later cloned into the GS115 strain of Pichia pastoris via pPIC3.5 vector. The expression of the GFP fusion cassette in P. pastoris was confirmed by fluorescence microscopy (Figure B). Interestingly, I am not able to visualize the expression of the GFP fusion cassette in P. pastoris in the same way as that of E.coli!
Why yeast cells do not show fluorescence recombinantly expressed GFP, in the same way, like that of bacterial cells?
Very nice images!
I guess the cell wall might make it harder to see the green color by eye on a plate. Usually, the greenish color is more visible in E. coli than it is in S. aureus, but I only know it for bacteria not for eukaryotes though.
I will like to say it is because of the pichia pastoris. Is it mainly yeast? It is not a bacteria? Yeast cells are more complex and require more work.
Hi Rahul,
how did you prepare the plates for Pichia? Using this system with the AOX1 promoter you would need to add methanol as inducer to the agar plates to get some possible GFP production on plate.
Also the production in P. pastoris might be lower if compared to E.coli. Did you use the exact same construct for both organisms or has the GFP been codon optimized for yeast production?
Best,
Pascal
I have expressed GFP fusions under Gal promoter in S. cerevisiae and the fluorescence is visible on gal pates (less clear than in Coli but still visible). I suspect it depends on the level of expression in your yeast.
Dear Pascal,
I have used a minimal methanol plate to check for GFP expression in P. pastoris and the GFP gene was synthesized and optimized for codon usage for expression in both E.coli and P. pastoris.
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