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Protein Preparation and Activity Identification of Rat Sentrin-specific Protease 1 Catalytic Domain |
Li MENG,Cai-ping DU**() |
Research Center for Biochemistry and Molecular Biology, Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical University, Xuzhou 221004, China |
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Abstract 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.
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Received: 26 April 2022
Published: 04 November 2022
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Corresponding Authors:
Cai-ping DU
E-mail: caipingdu@xzhmu.edu.cn
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