Construction and identification of bait plasmid carrying AMP-activated protein kinase ?2 in bacterial two-hybrid system
- VernacularTitle:AMP激活的蛋白激酶?2大肠杆菌双杂交诱饵重组质粒的构建及鉴定
- Author:
Qingying FU
;
Yuqi GAO
- Publication Type:Journal Article
- Keywords:
AMP-activated protein kinase;
bacterial two-hybrid;
bait plasmid
- From:Journal of Third Military Medical University
2003;0(19):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a bait plasmid in bacterial two-hybrid system.Methods A cDNA fragment encoding for rat AMP-activated protein kinase ?2(AMPK?2) was amplified by PCR and inserted into bacterial expression vector pBT.After confirmation with restricted endonuclease digestion and sequence analysis,bacterial reporter strain XL-1 Blue MRF' was transformed with pBT-AMPK?2 plasmid and the expression of the recombinant bait fusion protein was detected.To test whether the bait fusion protein had the capability of self-activation,the XL-1 Blue MRF' cells were cotransformed with the pBT-AMPK?2 plasmid and empty pTRG vector,and screened on 3-amino-1,2,4-triazole(3-AT) Selective Screening Medium plates.Results Restriction digestion and sequence analysis revealed that the AMPK?2 code sequence was correctly inserted into pBT with a right reading frame.pBT-AMPK?2 expressed ?cI/AMPK?2 fusion protein.Colonies were obtained on no 3-AT Nonselective Screening Medium plates when XL-1 Blue MRF' cells were cotransformed with recombinant pBT-AMPK?2 and empty pTRG vector,while none grew on 3-AT plates,indicating that the recombinant plasmid pBT-AMPK?2 expressed AMPK?2/?cI fusion protein correctly,and was incapable of activation of the reporter cassette in the absence of an interaction partner.Conclusion The recombinant plasmid pBT-AMPK?2 could be used as "bait plasmid" to screen cDNA library.
