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| Screening and identification of candidate proteins interacting with lung cancer metastasis-related protein 1 of human 95D cell line cDNA library by yeast two hybridization |
| LI Yanqin, LIU Yang, LIANG Liling, and XU Yang. |
| Department of Respiratory Medicine, PLA General Hospital, Beijing 100853, China |
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Abstract Objective To screen and identify the proteins that interact with LCMR1 from human 95D cell line library by the yeast two-hybrid system.Methods The bait plasmid, pGBKT7-LCMR1 , which expressed LCMR1 fusion protein, was transformed into AH109 yeast strains before yeast two-hybrid screening was performed by mating AH109 with Y187 containing human 95D cell cDNA library plasmids. SD/-Ade/-His/-Leu/-Trp medium and X-α-Gal were used for selecting and screening the interacting proteins.Moreover, back-cross was performed to confirm the positive clones .The vectors of Myc-tagged fusion protein and flag-tagged interacting proteins were constructed, identified and cotransfected into human embryo kidney 293 cells.The interactions between them were investigated by the immunoprecipitation(CO-IP) and GST pull-down system.Results The bait plasmid pGBKT7-LCMR1 was identified and transformed into AH109 and six true positive proteins with known functions were obtained through sequencing and bioinformatics analysis, which were GNG5, RPL29, DEK, CCAR1, G3BP1 and LGMN. GNG5 and RPL29 were selected for further study. The eukaryotic expression vectors of pcDNA3.0-flag -RPL29, pcDNA3.0 -flag-GNG5 and pCMV-Myc-LCMR1 were constructed and confirmed with double restriction enzyme digestion and co-transfected into 293 cell line.The interactions between RPL29 and LCMR1 were identified with Western blot after Co-IP, andno interactions between GNG5 and LCMR1 were detected.The same results were found in the subsequent experiment of GST pull down.Conclusions Proteins in 95D cell line library that can interact with LCMR1 are successfully screened out and identified.The interactions between LCMR1 and RPL29 are confirmed by the CO-IP and GST pull-down system.
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Received: 24 February 2018
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2018, Vol. 29 
