Binding Of DOC-1R With CK2? And PRDX1 Proteins And The Preliminary Analysis Of The Binding Regions

Object:Our previous data obtained from Mass Spectrometry(MS)suggested that Deleted In Oral Cancer-1 Related(DOC-1R)might interact with ? subunit of Casein Kinase 2(CK2)and peroxiredoxin 1(PRDX1).To confirm it,Pull-Down and Co-Ip assays were applied.Furthermore,the interacting regions of DOC-1R with CK2? and PRDX1 were investigated with recombinant technology and Pull-Down assays.The foundation was laid for further functional studies of DOC-1R.Methods:1 The interactions of DOC-1R with CK2? and PRDX1 were detected by Pull-Down assaysThe GST and recombinant full-length GST-DOC-1R proteins were prepared from E.coli BL21 inducing by IPTG.Moreover,Pull-Down assays were used and the interactions of DOC-1R with CK2? and PRDX1 were identified by Western Blot,respectively.2 The interactions of DOC-1R with CK2? and PRDX1 were detected by Co-IpHeLa cells infected with Lentivirus vectors were prepared previously.Co-Ip assays were applied in the cells.The interactions of DOC-1R with CK2? and PRDX1 were detected by Western Blot,respectively.3 Construction of truncated prokaryotic expression vectors of DOC-1R,and the interacting regions of DOC-1R with CK2? and PRDX1 were investigated by GST Pull-Down assaysTruncated DOC-1R prokaryotic expression vectors lacking coding regions of 1-42,85-126,1-63 were constructed using recombinant technology,respectively.Colony PCR,restriction enzyme digestion and DNA sequencing were used to identify target vectors.The three pGEX-DOC-1R deleted recombinant vectors were transformed into E.coli BL21 competent cells.The expressions of deleted DOC-1R were induced by IPTG and confirmed by SDS-PAGE gel electrophoresis and Coomassie blue stain.In addition,Pull-Down assay were conducted,respectively.Results:1 The interactions of DOC-1R with CK2? and PRDX1 were confirmed by Pull-Down.The interactions between DOC-1R with CK2? and PRDX1 were detected under theexperimental conditions,respectively.Additionally,the regulatory subunit of CK2?,CK2?,was identified in the elution mix.2 The interactions of DOC-1R with CK2? and PRDX1 were detected by Co-Ip.The interactions between DOC-1R with CK2? and PRDX1 in HEK-293 were confirmed,respectively.3 Construction of truncated prokaryotic expression vectors of DOC-1R,and the interacting regions of DOC-1R with CK2? and PRDX1 were investigated by GST Pull-Down.DNA sequencing analysis showed that the truncated DOC-1R DNA fragments were successfully inserted into pGEX-5X-1 vectors.The sequences of the target fragments and the open reading frames were correct which demonstrated the construction of truncated DOC-1R vectors were successful.Furthermore,the truncated DOC-1RdelN42,DOC-1RdelC42,and DOC-1RdelN63 proteins were abundantly expressed at 37°C after the addition of IPTG for 3 h.Under the conditions,the 107-119 amino acid region of the DOC-1R interacted with CK2? and the 85-126 amino acid region of the DOC-1R interacted with PRDX1.Conclusion:1.The interactions between DOC-1R with CK2? and PRDX1 were detected,respectively.2.The 107-119 amino acid region of DOC-1R interacted with CK2?,and the 85-126 amino acid region of DOC-1R interacted with PRDX1.