Testing interaction between two know proteins by ras recruitment system / 重庆医科大学学报

ABSTRACT

Objective:Introduce yeast two-hybrid system RRS screening system through interaction between two known proteins,Pak and Chp,in order to apply this system on research of the interaction between other proteins.Methods:Select the yeast strain cdc25-2 for single clone that displays low reversion rate,and prepare the competences of the yeast by LiAc.Only transform the plasmid Met425-Myc-Ras-Pak into the yeast to eliminate its self-activation.Co-transform the plasmids Met425-Myc-Ras-Pak and Ura-M-ChpAc into the yeast,set up some controls,observe their growth on different mediums at 36℃.Results:The cdc25-2 cells which are used to prepare competences have no reversion.After the transformation of the plasmid Met425-Myc-Ras-Pak into the yeast,there is neither toxicity nor self-activation.Only when the two plasmids which can express the two corresponding known proteins are co-transformed,and only on the medium suitable for their expression,the cdc25-2 cells can grow at 36℃.Conclusion:(1)The RRS screening system verifies the interaction between Pak and Chp correctly.So it can be used for research of the interaction between other known proteins.(2)We can construct a bait of an known protein,then use the RRS screening system to look for the interacted proteins.