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

Abstract

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 twohybrid experiments and was confirmed to be interacted with TSCBP86-IV in human sperm cells. K E Y W O R D S phosphoglycerate kinase 2, protein interaction, spermatozoa, testis-specific calcium-binding protein CBP86-IV

Comprehensive Profiles of mRNAs and miRNAs Reveal Molecular Characteristics of Multiple Organ Physiologies and Development in Pigs

“…ULK2 , when knocked down, will inhibit swine Sertoli cell autophagy (Ran et al, 2018a). Meanwhile the testis-specific genes identified in this study such as SPEM1 , TNP1 , PRM1 , DAZL (Hashemi et al, 2018), and CABYR (Shen et al, 2019) were also published as specific expression in human or mouse. As we know, tissue-specific functions are a result of specific expression and regulation of genes across an organism’s lifespan.…”