Howdy, I have been trying to express a big protein (~1500 AAs) of bacterial origin in baker's yeast which needs to get into the nucleus. The protein is fused to E2-crimson fluorescence protein, and SV40 NLS is used. But fluorescence microscopy revealed that only a small fraction (~10%) of yeast cells had red signals in nucleus. The picture is attached.
I wonder why nuclear localization is so inefficient, and any suggestions to improve are welcome!!
Thanks!