Abstract: Objective To identify whether human TRIM69 could catalyze formation of multiubiquitinylated products. Methods The gene of human TRIM69 was cloned into a procaryotic expression vector, which was then transformed into E.coli. Purified protein of TRIM69 was obtained through the process of induction, expression, and purification, which was used in the following experiment of ‘in vitro ubiquitination assay’ to detect whether TRIM69 could catalyze formation of multiubiquitinylated products. In addition, we constructed TRIM69 gene full length or a point mutagenesis of RING domain sequence or a deleted RING domain sequence into a eukaryotic expression vector. Then the corresponding vectors were transiently transfected into HEK293T cells or HeLa cells of stably expressed ubiquitination. The experiment of immunoprecipitation combined with immunoblotting was performed to detect whether TRIM69 could catalyze formation of multiubiquitinylated products in cells. Results The ubiquitination assay results demonstrated that TRIM69 could catalyze formation of multiubiquitinylated products, which was dependent on its RING domain. Conclusion The enzyme activity of TRIM69 in catalyzing the formation of muti-uiquitinylated products was successfully identified, which builds up a basis for further characterizing TRIM69 as a novel E3 ubiquitin ligase.

Key words: spermatogenesis, E3 ubiquitin ligase, TRIM, RING finger