LAUSR

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.