[Construction and verification of bait protein RGS4-fusion expression plasmid in yeast two-hybrid system].
OBJECTIVE
To research the interaction between glucocorticosteroid receptor domains and regulator of G protein signaling 4 (RGS4) protein.


METHODS
The RGS4 gene was used as the bait protein gene to construct the fusion bait expression plasmid of yeast two-hybrid. The whole encoding sequence of RGS4 gene was amplified by RT-PCR method. With confirmed by electrophoresis, the RGS4 gene was cloned into the MCS of the plasmid pGBKT7 to form a recombinant plasmid pGBKT7-RGS4 and the sequence of the recombinant plasmid was sequenced. According to the protocol of yeast two-hybrid system gold III, the competent Y187 yeast was prepared and transformed with recombinant plasmid pGBKT7-RGS4. Following that, the toxicity and autonomous activation of this recombinant plasmid pGBKT7-RGS4 in Y187 yeast were tested. In the end, we verified the normal expression of the RGS4 fusion protein in vitro by TNT system and in vivo by Weston blot.


RESULTS
The sequence of the recombinant plasmid was verified to be correct, as compared with the sequence provided by GenBank. The protein could be correctly synthesized in vivo, and no autonomous activation and toxicity was observed in Y187 yeast.


CONCLUSIONS
The recombinant plasmid may be used as the fusion bait plasmid in yeast two-hybrid system III, and the recombinant RGS4 fusion protein can be used as the bait protein successfully.
Li
Ping
P
Research Institute of Field Surgery, the Third Military Medical University, Chongqing 400042, China.
Wang
Xia
X
Zhou
Yuan-Guo
YG
Chen
Xing-Yun
XY
D000818:Animals; D015870:Gene Expression; D010957:Plasmids / Q000235:genetics*; D017434:Protein Structure, Tertiary; D020710:RGS Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014162:Transfection; D020798:Two-Hybrid System Techniques
D020710:RGS Proteins; D011993:Recombinant Fusion Proteins; C098799:RGS4 protein