Testis-specific calcium-binding protein CBP86-IV (CABYR) binds with phosphoglycerate kinase 2 in vitro and in vivo experiment

The investigation of the interacting proteins with testis-specific calcium-binding protein CBP86-IV (CABYR) was carried out in human spermatozoa. The total RNA from human spermatozoa was extracted, and the ORF sequence of TSCBP86-IV gene was amplified and cloned into expression vector pET-28a. The positive Recombinant clones were transformed into Escherichia coli strain BL21 (DE3) to express fusion protein. Then, co-immunoprecipitation (Co-IP) of TSCBP86-IV was performed in BL21 cell lysate expressing CBP86-IV Recombinant protein. The immune complex was captured and identified by mass spectrometry. Reverse Co-IP of potential interacting proteins was performed in human sperm cell lysate. The potential protein interactions were confirmed by yeast two-hybrid system. Thirteen proteins were successfully identified in immune complex from E. coli cell lysate. Phosphoglycerate kinase 2 (PGK2) further showed positive results both in reverse Co-IP and yeast two-hybrid experiments and was confirmed to be interacted with TSCBP86-IV in human sperm cells.