原核类泛素-蛋白酶体系统（Pup-proteasome system，PPS）是原核生物中一种重要的蛋白降解途径，具有重要的生理功能。PafA（proteasome accessory factor A）是Pup-蛋白酶体系统中的一个辅助因子，参与催化Pup与底物的Lys位点的连接。PafA的N端结构域较大，酶的活性中心和ATP结合位点均位于N端结构域。其C端结构域较小，功能尚不清楚，我们猜测其直接或间接参与了底物识别过程。本次实验我们在E.coli中转化WT及C端突变（LI-SS）的PafAcglu重组质粒并诱导表达，纯化PafAcglu及其结合蛋白，对比分析C端突变是否会使PafA结合蛋白存在差异。结果显示PafA能和E.coli中一些常见的分子伴侣和核糖体亚基结合，与PafA(WT)相比，PafA(LI-SS)的结合蛋白数量明显变少，SlyD、GroEL等结合蛋白能与PafA(WT)共纯化，却不能与PafA(LI-SS)共纯化，说明PafA确实可能通过与分子伴侣等蛋白结合来完成底物识别过程。

Prokaryotic ubiquitin-like proteasome system is an important protein degradation pathway in bacterial and has essential physiological functions. PafA (proteasome accessory factor A) is an cofactor in PPS, catalyzing the formation of isopeptide bond between Pup and specific lysine of the substrate. The N-terminal domain of PafA is larger, and the active center and ATP-binding site of the enzyme are all located here. The C-terminal domain is small and its function is not clear yet. We speculate that it is directly or indirectly involved in the process of substrate recognition. In this study we transfer the recombinant plasmid of wild type and C-terminal mutant PafAcglu in E.coli, and induce them to express. Then we purify PafAcglu and its binding proteins, and compare them to determine whether the C-terminal mutation would make a difference in the PafA binding protein. The results showed that PafA can bind to some common chaperone and ribosomal subunits in E. coli. Compared with PafA (WT), the number of binding proteins of PafA (LI-SS) is significantly reduced. SlyD and GroEL can be co-purified with PafA(WT) but can’t be co-purified with PafA (LI-SS), suggesting that maybe PafA can finish the process of substrate recognition by combining with proteins such as molecular chaperones.