大鼠SUMO特异性蛋白酶1催化区蛋白制备及功能鉴定*

目的: 制备大鼠SUMO特异性蛋白酶1(sentrin-specific protease,SENP1)催化结构域(SENP1C)蛋白,并鉴定其酶活性。方法: 分别以大鼠SENP1-pcDNA3.1和EGFP-pcDNA3.1重组体为模板,PCR扩增目的基因,克隆入pGEM-T载体;酶切鉴定后,再亚克隆入原核表达载体pET-28a;阳性重组体导入原核表达细胞BL-21,异丙基硫半乳糖苷(IPTG)诱导蛋白质表达;SDS-PAGE及考马斯亮蓝染色鉴定蛋白质的表达。Ni-NTA吸附纯化蛋白质并透析处理,SDS-PAGE及考马斯亮蓝染色鉴定蛋白质的纯度;1 μmol/L及5 μmol/L Tat-EGFP分别孵育HT22细胞不同时间,荧光显微镜下观察细胞转染情况。采用5 μmol/L Tat-SENP1C预孵育HT22细胞10 h,免疫印迹检测整体蛋白质的SUMO化水平;用5 μmol/L Tat-SENP1C预孵育HT22细胞或过表达Myc-Akt1和HA-SUMO1的HT22细胞10 h后,免疫沉淀和免疫印迹检测内源性和外源性Akt1与SUMO1的结合(SUMO化)。结果: Tat-SENP1C-pET-28a和Tat-EGFP-pET-28a重组原核表达载体成功构建,IPTG可以诱导蛋白质高表达;采用Ni-NTA纯化和透析可获得较高纯度的蛋白质;5 μmol/L Tat-EGFP孵育HT22 细胞10 h后,蛋白质穿膜效率较高;Tat-SENP1C重组蛋白可以显著降低HT22细胞中整体蛋白质的SUMO化以及内源性和外源性Akt1 SUMO化。结论: Tat-SENP1C-pET-28a和Tat-EGFP-pET-28a重组原核表达载体构建成功,且被IPTG诱导后可高效表达蛋白质;纯化的Tat-SENP1C蛋白具有较强的穿膜能力及酶活性。

Protein Preparation and Activity Identification of Rat Sentrin-specific Protease 1 Catalytic Domain

Objective: Preparation and enzymatic activity identification of sentrin-specific protease1 (SENP1) catalytic domain (SENP1C). Methods: The target genes were amplified by PCR from SENP1-pcDNA3.1 and EGFP-pcDNA3.1, and then cloned into pGEM-T vector. After enzyme digestion, the digested cDNAs were then subcloned into the prokaryotic expression vector pET-28a. Next, the positive recombinants were transfected into prokaryotic expression cells BL-21, which were then induced by isopropyl thiogalactoside (IPTG). The protein expression was identified by SDS-PAGE and coomassie brilliant blue staining. The extracted proteins were purified by Ni-NTA and dialysis treatment, and the protein purity was further checked by SDS-PAGE and coomassie brilliant blue staining. HT22 cells was pre-incubated with 1 μmol/L or 5 μmol/L Tat-EGFP for different times, and cell transfection was observed by fluorescence microscope. After pretreatment with 5 μmol/L Tat-SENP1 for 10 h, the SUMOylation of overall protein in HT22 cells was detected by immunoblot analysis. In addition, immunoprecipitation and immunoblotting were used to evaluate endogenous and exogenous Akt1-SUMO1 conjugations in HT22 cells or HT22 cells overexpressing Myc-Akt1 and HA-SUMO1. Results: Tat-SENP1C-pET-28a and Tat-EGFP-pET-28a prokaryotic expression recombinants were successfully constructed, which were efficiently induced to express target proteins by IPTG. The high purity target proteins were obtained by Ni-NTA and dialysis. After incubation with 5 μmol/L Tat-EGFP for 10 h, the penetration efficiency was higher in HT22 cells. Tat-SENP1C reduced the levels of total SUMOylation and endogenous and exogenous Akt1-SUMO1 conjugations significantly. Conclusion: Tat-SENP1C-pET-28a and Tat-EGFP-pET-28a prokaryotic expression recombinants were successfully constructed, and can be highly induced to express target proteins by IPTG. The purified Tat-SENP1C retains strong membrane penetration ability and enzymatic activity.