Screening Of Proteins Interacted With MEKK2

Study of interacting proteins is the most important method to investigate the function of genes.As the development of the proteomics,large-scale screening interacting proteins in different biology system became more and more important. Most biology function is finished by protein complex.To date,yeast two hybrid and tandem affinity purification are the common methods to screen interacting protein.MEKK2 is a Ser/Thr protein kinase belonging to MA3P family.MEKK2 is expressed in multiple tissues,especially in the spleen and peripherals,but its physiological functions in different tissues are largely unknown.In vitro studies show that MEKK2 activates the JNK1/2,ERK1/2,p38 and ERK5 MAPKs mediated by MEK1/2,MKK3/6,MEK5,respectively.Consistent with this finding,MEKK2 activate transcription factor NF-κB and AP-1,and plays important role in the PKC-2 signal pathway too.The aim of the present study was to screening MEKK2-interacting protein by yeast two hybrid and tandem affinity purification use the full-length MEKK2 as bait. In the yeast two hybrid studies,we cloned the MEKK2 into pGBKT7 vector.The bait and 293 cell and B cell library were transfeeted into AH109 yeast cells through PEG/LiAC method,screening positive clones in the nutrition limitation culture.The results indicate that yeast two hybrid is not suitable in the practical screening of MEKK2-interacting proteins,as there were too many clones(around 2000 clones) in the 4- plate.In the TAP study,we chose SBP and CBP tag.MEKK2 was cloned into pCTAP vector and transfected into HEK293 cells with pSUPER,based on the puromycin to construct stable cell lines.Two-step affinity purification enables the isolation of protein complexes under close-to-physiological conditions and analyzed by mass spectrometry.216 proteins were obtained in total.Among those proteins, Mip1,MEK3,MEK6 were previously identified interacting proteins,50 were non-speicific proteins and 163 were possible interacting proteins,respectively.The further identification and characterization of 163 possible interacting proteins remain to be studied.