Characterization Of LOX Interactome By Tandem Affinity Purification Strategy Followed By LC-MS/MS In Breast Cancer Cell

OBJECTIVE: in order to further screening and research the function of lysyloxidase(LOX) and its interaction proteins in MDA-MB-231invasive breast cancer cell line, usingtandem affinity purification by gateway to lentiviral vector caring recombination LOX fusion withStrepII/FLAG tag,the LOX complex was purified by the tandem affinity purification andidentified by LC-MS/MS.METHODS: To gain the vector of LOX-SF recombination protein by polymerase chainreaction, adopting overlap PCR to construct the fusion gene through a chimeric primer, then therecombination vector GV303/LOX-SF was constructed by the fusion gene and GV303weredigested. To packaging lentivirus vector from293T cells using three-plasmid transienttransfections, then MDA-MB-231cells were transfection by recombinant lentivirus vector, Thecell line stably expression of fusion protein preliminary detected by realtime fluorescencequantitative PCR and Western Blot. The cells were lysed and LOX complex were purified throughthe Streptavidin beads and the anti-FLAD M2agorose beads. The complex were separated bySDS-PAGE, silver stained bands were excised and digested in gel, then identified by(Nano-RPLC)/ESI-MS/MS.RESULT: Construction of the lentivirus expression vector that carrying tandem affinitypurification tag, The MDA-MB-231cells stably expression LOX-SF recombination protein thatdetected by anti-Tag, then purify the LOX-SF protein through the tandem affinity purification(TAP) method. Totally,425proteins identified at95%confidence level by (Nano-RPLC)/ESI-MS/MS.CONCLUSION: In the425of identified proteins, There were20associated network functions theLOX involved mention in the analysis result, most of the functions are related with Cancer,Infectious Disease, Hematological Disease, Immunological Disease and so on. These protein thatwere identified in mTOR Signaling、Remodeling of Epithelial Adherens Junctions、ActinCytoskeleton Signaling at the front top of Canonical pathways, which indicate LOX mightinvolved in tumor invasion and metastasis. Protein interactome divided into16networks by themolecular function on the database, which including LOX directly involved in. The successfullyexpression and purification of LOX fusion with SF tag in MDA-MB-231cells because of the recombinant vector GV303/LOX-SF was constructed, laying the foundation to further study theprotein-protein interactions of LOX in breast cancer cell lines.