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

doi:10.13523/j.cb.2204069

China Biotechnology

2022, Vol. 42

Issue (10): 31-38 DOI: 10.13523/j.cb.2204069

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

Download:

HTML

PDF(2038KB) HTML
Export: BibTeX | EndNote (RIS)

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.

Key words： Sentrin-specific protease 1 Catalytic domain Prokaryotic expression vector deSUMOylation Akt1

Received: 26 April 2022 Published: 04 November 2022

ZTFLH:

Q786

Corresponding Authors: Cai-ping DU E-mail: caipingdu@xzhmu.edu.cn

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	Li MENG
	Cai-ping DU

Cite this article:

Li MENG,Cai-ping DU. Protein Preparation and Activity Identification of Rat Sentrin-specific Protease 1 Catalytic Domain. China Biotechnology, 2022, 42(10): 31-38.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2204069 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I10/31

Table 1 Primers for Tat-SENP1C and Tat-EGFP

Fig.1 Construction of Tat-SENP1C and Tat-EGFP prokaryotic expression vector (a) Identification of Tat-SENP1C and Tat-EGFP PCR products (b) Tat-SENP1C and Tat-EGFP cloned to pGEM-T vector separately (c) Tat-SENP1C-T vector and Tat-EGFP-T vector were digested by NcoI and NotI (d) Tat-SENP1C or Tat-EGFP subcloned to pET-28a (e) Tat-SENP1C-pET-28a and Tat-EGFP-pET-28a recombinants were digested by NcoI and NotI. 1, 2: The recombinants vector failed to digested by NcoI and NotI;M: DNA marker

Fig.2 Examination of Recombinant protein expression by Comassie blue staining

Fig.3 Identification of the Tat-SENP1C and Tat-EGFP purity (a) Comassie blue staining results of purified Tat-SENP1C; (b) Comassie blue staining results of purified Tat-EGFP

Fig.4 Identification of Tat-EGFP penetration efficiency

Fig.5 Detection of Tat-SENP1C enzymatic activity (a) Effect of Tat-SENP1C on the total protein SUMOylation in HT22 cells. The SUMOylation level of overall protein were detected by immunoblotting with anti-SUMO1 antibody. Actin served as an internal control (b) Effect of Tat-SENP1C on the endogenous Akt1 SUMOylation. The Akt1-SUMO1 conjugation was checked by immunoprecipitation (IP) with anti-SUMO1 antibody followed by immunoblot (IB) with anti-Akt1 antibody. Akt1 expression was detected by immunoblot with anti-Akt1 antibody. Control: Unincubated Tat protein group; ^* P<0.05 vs control group (c) Effect of Tat-SENP1C on the exogenous Akt1 SUMOylation. The Akt1-SUMO1 conjugation and Akt1 protein expression were checked by immunoprecipitation (IP) with anti-Akt1 antibody followed by immunoblot (IB) with anti-SUMO1 or anti-Akt1 antibodies individually. Control: Unincubated Tat protein group; ^* P<0.05 vs control group


[1]	Hay R T. SUMO: a history of modification. Molecular Cell, 2005, 18(1): 1-12. pmid: 15808504

[2]	Bailey D, O’Hare P. Characterization of the localization and proteolytic activity of the SUMO-specific protease, SENP1. Journal of Biological Chemistry, 2004, 279(1): 692-703. doi: 10.1074/jbc.M306195200 pmid: 14563852

[3]	Kolli N, Mikolajczyk J, Drag M, et al. Distribution and paralogue specificity of mammalian deSUMOylating enzymes. The Biochemical Journal, 2010, 430(2): 335-344. doi: 10.1042/BJ20100504

[4]	Xu Z, Au S W N. Mapping residues of SUMO precursors essential in differential maturation by SUMO-specific protease, SENP1. The Biochemical Journal, 2005, 386(Pt 2): 325-330. doi: 10.1042/BJ20041210

[5]	Hickey C M, Wilson N R, Hochstrasser M. Function and regulation of SUMO proteases. Nat Rev Mol Cell Biol. 2012, 13(12):755-766. doi: 10.1038/nrm3478

[6]	Nayak A, Müller S. SUMO-specific proteases/isopeptidases: SENPs and beyond. Genome Biology, 2014, 15(7): 422. doi: 10.1186/s13059-014-0422-2 pmid: 25315341

[7]	Kunz K, Piller T, Müller S. SUMO-specific proteases and isopeptidases of the SENP family at a glance. Journal of Cell Science, 2018, 131(6): jcs211904.

[8]	He J L, Shangguan X, Zhou W, et al. Glucose limitation activates AMPK coupled SENP1-Sirt 3 signalling in mitochondria for T cell memory development. Nature Communications, 2021, 12: 4371. doi: 10.1038/s41467-021-24619-2

[9]	Shao L, Zhou H J, Zhang H F, et al. SENP1-mediated NEMO deSUMOylation in adipocytes limits inflammatory responses and type-1 diabetes progression. Nature Communications, 2015, 6: 8917. doi: 10.1038/ncomms9917 pmid: 26596471

[10]	Yamaguchi T, Sharma P, Athanasiou M, et al. Mutation of SENP1/SuPr-2 reveals an essential role for desumoylation in mouse development. Molecular and Cellular Biology, 2005, 25(12): 5171-5182. pmid: 15923632

[11]	Sun M L, Chen X, Yin Y X, et al. Role of pericyte-derived SENP 1 in neuronal injury after brain ischemia. CNS Neuroscience & Therapeutics, 2020, 26(8): 815-828.

[12]	Zhang H J, Wang Y, Zhu A X, et al. SUMO-specific protease 1 protects neurons from apoptotic death during transient brain ischemia/reperfusion. Cell Death & Disease, 2016, 7(11): e2484.

[13]	Shen L N, Tatham M H, Dong C J, et al. SUMO protease SENP 1 induces isomerization of the scissile peptide bond. Nature Structural & Molecular Biology, 2006, 13(12): 1069-1077. doi: 10.1038/nsmb1172

[14]	Xu Z, Chau S F, Lam K H, et al. Crystal structure of the SENP 1 mutant C603S-SUMO complex reveals the hydrolytic mechanism of SUMO-specific protease. The Biochemical Journal, 2006, 398(3): 345-352. doi: 10.1042/BJ20060526

[15]	孟利, 杜彩萍. 大鼠His-Akt1重组蛋白的真核表达、蛋白纯化及活性鉴定. 生物技术通报, 2020, 36(12): 98-103. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0497

[15]	Meng L, Du C P. Eukaryotic expression, purification and activity identification of rat his-Akt1 recombinant protein. Biotechnology Bulletin, 2020, 36(12): 98-103. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0497

[16]	Du C P, Wang M, Geng C, et al. Activity-induced SUMOylation of neuronal nitric oxide synthase is associated with plasticity of synaptic transmission and extracellular signal-regulated kinase 1/2 signaling. Antioxidants & Redox Signaling, 2020, 32(1): 18-34.

[17]	Li R, Wei J, Jiang C, et al. Akt SUMOylation regulates cell proliferation and tumorigenesis. Cancer Research, 2013, 73(18): 5742-5753. doi: 10.1158/0008-5472.CAN-13-0538 pmid: 23884910

[18]	韦雪芳, 王冬梅, 刘思, 等. 信号肽及其在蛋白质表达中的应用. 生物技术通报, 2006(6): 38-42.

[18]	Wei X F, Wang D M, Liu S, et al. Signal sequence and its application to protein expression. Biotechnology Bulletin, 2006(6): 38-42.

[19]	Wang F H, Wang Y, Zhang X, et al. Recent progress of cell-penetrating peptides as new carriers for intracellular cargo delivery. Journal of Controlled Release, 2014, 174: 126-136. doi: 10.1016/j.jconrel.2013.11.020 pmid: 24291335

[20]	Wu B H, Li M S, Li K K, et al. Cell penetrating peptide TAT-functionalized liposomes for efficient ophthalmic delivery of flurbiprofen: penetration and its underlying mechanism, retention, anti-inflammation and biocompatibility. International Journal of Pharmaceutics, 2021, 598: 120405. doi: 10.1016/j.ijpharm.2021.120405

[21]	Yau T Y, Molina O, Courey A J. SUMOylation in development and neurodegeneration. Development (Cambridge, England), 2020, 147(6): dev175703.

[22]	Buccarello L, Dragotto J, Iorio F, et al. The pivotal role of SUMO-1-JNK-Tau axis in an in vitro model of oxidative stress counteracted by the protective effect of curcumin. Biochemical Pharmacology, 2020, 178: 114066. doi: 10.1016/j.bcp.2020.114066

[23]	Yang T Y, Sun J Y, Wei B, et al. SENP1-mediated NEMO de-SUMOylation inhibits intermittent hypoxia induced inflammatory response of microglia in vitro. Journal of Cellular Physiology, 2020, 235(4): 3529-3538. doi: 10.1002/jcp.29241

[24]	Wang H W, Yang T Y, Sun J Y, et al. SENP1 modulates microglia-mediated neuroinflammation toward intermittent hypoxia-induced cognitive decline through the de-SUMOylation of NEMO. Journal of Cellular and Molecular Medicine, 2021, 25(14): 6841-6854. doi: 10.1111/jcmm.16689

[1]	LIU Hai-Yan- Gu-Yu-Chao- Fu-Wen-Xi- Xu-Wen-Gong. Akt1 Silencing Inhibits Lung Metastasis of Murine Breast Cancer Cells[J]. China Biotechnology, 2009, 29(03): 14-19.

Viewed

Full text

Abstract

Cited

Shared

Discussed