Prokaryotic Expression，Purification and Crystallization of N-terminal Domain of Nucleocapsid Protein in SARS-CoV-2

doi:10.13560/j.cnki.biotech.bull.1985.2022-0400

Abstract

Abstract:

The expression and purification of the N-terminal domain of SARS-CoV-2 nucleocapsid protein（N-protein）were explored through prokaryotic expression system, and its growth conditions were optimized, aiming to provide a basis for structural biological research of SARS-CoV-2 nucleocapsid protein. The N-terminal domain gene sequence of the N protein was synthesized, the prokaryotic expression vector was constructed, and the recombinant target protein was expressed by Escherichia coli. The expressed products were purified by Ni²⁺ affinity chromatography and gel filtration chromatography. The purity of the recombinant protein was identified by SDS-PAGE electrophoresis. The prokaryotic recombinant vector pET28b-N-N was successfully constructed. E. coli was amplified and grew at 37℃ and induced to express at 16℃，and 80% of the target protein was expressed in the form of soluble protein. The purity of the target protein was >90% via protein glue integral analysis and obtaining the integral ratio by Quantity One software after Ni²⁺ affinity chromatography and gel filtration chromatography. The protein crystal was screened by Hampton protein crystallizer kit, and a high quality crystal of the N-terminal of the N-protein was obtained. Electrophoretic Mobility Shift Assay（EMSA）was used to qualitatively validate the definite interaction between the N-terminal of the N-protein with the specific RNA fragments, and confirmed the function of the N-terminal domain of N-protein stabilizing the SARS-CoV-2 genome. The results provide basis for the 3D structure of the N-terminal of the N-protein and the subsequent antibody development.

Key words: SARS-CoV-2, nucleocapsid protein, purification, crystallization

CHEN Duo, LIU Yong-zhe. Prokaryotic Expression，Purification and Crystallization of N-terminal Domain of Nucleocapsid Protein in SARS-CoV-2[J]. Biotechnology Bulletin, 2022, 38(12): 149-155.

Figures/Tables 6

Table 1 Primer sequences

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
N-N-F	GGAATTCCATATGAATAATACTGCGTCTTGGTT
N-N-R	CCGCTCGAGTTACCCTTCTGCGTAGAAGCCTT

Fig. 1 Comparison of N protein amino acid sequences of SARS-CoV-2，SARS-CoV and MERS-CoV

Fig. 2 PCR amplification result of N-N protein 1：PCR amplification product. M：DNA molecular weight standard

Fig. 3 Expression and purification of N-N protein A：The N-N protein samples were purified by Superdex 75 column. B：The purified N-N protein samples were analyzed on SDS-PAGE. M：Protein marker. 1-3：The N-N protein；4：Before-induced.5：After-induced

Fig. 4 EMSA result of binding of N-N protein and RNA 1：Normal RNA control. 2-7：Incubation of RNA and N-N protein（The N-N protein concentrations correspond to 1，2，3，4，5 and 6 mg/mL respectively）

Fig. 5 Optimization of N protein crystal A：Making Initial crystal by PEG/Ion Screen 23. B：Making further crystal by adding Crystal Screen 45. C：Making final crystal by adding Additive Screen 25

References 30

[1]	Dong ES, Du HR, Gardner L. An interactive web-based dashboard to track COVID-19 in real time[J]. Lancet Infect Dis, 2020, 20(5):533-534. doi: S1473-3099(20)30120-1 pmid: 32087114
[2]	Huang CL, Wang YM, Li XW, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China[J]. Lancet, 2020, 395(10223):497-506. doi: S0140-6736(20)30183-5 pmid: 31986264
[3]	Anand KB, Karade S, Sen S, et al. SARS-CoV-2:camazotz’s curse[J]. Med J Armed Forces India, 2020, 76(2):136-141. doi: 10.1016/j.mjafi.2020.04.008 URL
[4]	Gordon DE, Jang GM, Bouhaddou M, et al. A SARS-CoV-2 protein interaction map reveals targets for drug repurposing[J]. Nature, 2020, 583(7816):459-468. doi: 10.1038/s41586-020-2286-9 URL
[5]	Romano M, Ruggiero A, Squeglia F, et al. A structural view of SARS-CoV-2 RNA replication machinery:RNA synthesis, proofreading and final capping[J]. Cells, 2020, 9(5):1267. doi: 10.3390/cells9051267 URL
[6]	Hu B, Guo H, Zhou P, et al. Characteristics of SARS-CoV-2 and COVID-19[J]. Nat Rev Microbiol, 2021, 19(3):141-154. doi: 10.1038/s41579-020-00459-7 pmid: 33024307
[7]	Yang D, Leibowitz JL. The structure and functions of coronavirus genomic 3' and 5' ends[J]. Virus Res, 2015, 206:120-133. doi: 10.1016/j.virusres.2015.02.025 pmid: 25736566
[8]	Uddin M, Mustafa F, Rizvi TA, et al. SARS-CoV-2/COVID-19:viral genomics, epidemiology, vaccines, and therapeutic interventions[J]. Viruses, 2020, 12(5):526. doi: 10.3390/v12050526 URL
[9]	Yao HP, Song YT, Chen Y, et al. Molecular architecture of the SARS-CoV-2 virus[J]. Cell, 2020, 183(3):730-738. e13. doi: 10.1016/j.cell.2020.09.018 pmid: 32979942
[10]	Ye QZ, West AMV, Silletti S, et al. Architecture and self-assembly of the SARS-CoV-2 nucleocapsid protein[J]. Protein Sci, 2020, 29(9):1890-1901. doi: 10.1002/pro.3909 URL
[11]	Peng Y, Du N, Lei YQ, et al. Structures of the SARS-CoV-2 nucleocapsid and their perspectives for drug design[J]. EMBO J, 2020, 39(20):e105938.
[12]	Jia Z, Liu C, Chen Y, et al. Crystal structures of the SARS-CoV-2 nucleocapsid protein C-terminal domain and development of nucleocapsid-targeting nanobodies[J]. FEBS J, 2021. Doi:10.1111/febs.16239.
[13]	张西西, 张怡青, 李玉林, 等. 新型冠状病毒(SARS-CoV-2)N蛋白C端重组蛋白的原核表达、纯化及应用[J]. 生物技术通报, 2021, 37(5):92-97. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0902 URL
	Zhang XX, Zhang YQ, Li YL, et al. Prokaryotic expression, purification and application of N protein C-terminal recombinant protein in novel coronavirus(SARS-CoV-2)[J]. Biotechnol Bull, 2021, 37(5):92-97.
[14]	Wu C, Qavi AJ, Moyle AB, et al. Domain-specific biochemical and serological characterization of SARS-CoV-2 nucleocapsid protein[J]. STAR Protoc, 2021, 2(4):100906. doi: 10.1016/j.xpro.2021.100906 URL
[15]	Zeng WH, Liu GF, Ma H, et al. Biochemical characterization of SARS-CoV-2 nucleocapsid protein[J]. Biochem Biophys Res Commun, 2020, 527(3):618-623. doi: 10.1016/j.bbrc.2020.04.136 URL
[16]	Gupta Y, Kumar S, Zak SE, et al. Antiviral evaluation of hydroxyethylamine analogs:Inhibitors of SARS-CoV-2 main protease(3CLpro), a virtual screening and simulation approach[J]. Bioorg Med Chem, 2021, 47:116393. doi: 10.1016/j.bmc.2021.116393 URL
[17]	Samper IC, McMahon CJ, Schenkel MS, et al. Electrochemical immunoassay for the detection of SARS-CoV-2 nucleocapsid protein in nasopharyngeal samples[J]. Anal Chem, 2022, 94(11):4712-4719. doi: 10.1021/acs.analchem.1c04966 pmid: 35263100
[18]	Low JS, Vaqueirinho D, Mele F, et al. Clonal analysis of immunodominance and cross-reactivity of the CD4 T cell response to SARS-CoV-2[J]. Science, 2021, 372(6548):1336-1341. doi: 10.1126/science.abg8985 pmid: 34006597
[19]	López-Muñoz AD, Kosik I, Holly J, et al. Cell surface SARS-CoV-2 nucleocapsid protein modulates innate and adaptive immunity[J]. bioRxiv, 2021. DOI:10.1101/2021.12.10.472169. doi: 10.1101/2021.12.10.472169 URL
[20]	Xie CZ, Ding HJ, Ding JZ, et al. Preparation of highly specific monoclonal antibodies against SARS-CoV-2 nucleocapsid protein and the preliminary development of antigen detection test strips[J]. J Med Virol, 2022, 94(4):1633-1640. doi: 10.1002/jmv.27520 URL
[21]	Li DD, Li JM. Immunologic testing for SARS-CoV-2 infection from the antigen perspective[J]. J Clin Microbiol, 2021, 59(5):e02160-e02120.
[22]	Wang YT, Long XY, Ding X, et al. Novel nucleocapsid protein-targeting phenanthridine inhibitors of SARS-CoV-2[J]. Eur J Med Chem, 2022, 227:113966. doi: 10.1016/j.ejmech.2021.113966 URL
[23]	Lucas C, Vogels CBF, Yildirim I, et al. Impact of circulating SARS-CoV-2 variants on mRNA vaccine-induced immunity[J]. Nature, 2021, 600(7889):523-529. doi: 10.1038/s41586-021-04085-y URL
[24]	Pitcovski J, Gruzdev N, Abzach A, et al. Oral subunit SARS-CoV-2 vaccine induces systemic neutralizing IgG, IgA and cellular immune responses and can boost neutralizing antibody responses primed by an injected vaccine[J]. Vaccine, 2022, 40(8):1098-1107. doi: 10.1016/j.vaccine.2022.01.025 URL
[25]	Chatzileontiadou DSM, Szeto C, Jayasinghe D, et al. Protein purification and crystallization of HLA-A 02:01 in complex with SARS-CoV-2 peptides[J]. STAR Protoc, 2021, 2(3):100635. doi: 10.1016/j.xpro.2021.100635 URL
[26]	Cubuk J, Alston JJ, Incicco JJ, et al. The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA[J]. Nat Commun, 2021, 12(1):1936. doi: 10.1038/s41467-021-21953-3 pmid: 33782395
[27]	Savastano A, Ibáñez de Opakua A, Rankovic M, et al. Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates[J]. Nat Commun, 2020, 11(1):6041. doi: 10.1038/s41467-020-19843-1 pmid: 33247108
[28]	Jack A, Ferro LS, Trnka MJ, et al. SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA[J]. PLoS Biol, 2021, 19(10):e3001425. doi: 10.1371/journal.pbio.3001425 URL
[29]	Parashar NC, Poddar J, Chakrabarti S, et al. Repurposing of SARS-CoV nucleocapsid protein specific nuclease resistant RNA aptamer for therapeutics against SARS-CoV-2[J]. Infect Genet Evol, 2020, 85:104497. doi: 10.1016/j.meegid.2020.104497 URL
[30]	Tatar G, Ozyurt E, Turhan K. Computational drug repurposing study of the RNA binding domain of SARS-CoV-2 nucleocapsid protein with antiviral agents[J]. Biotechnol Prog, 2021, 37(2):e3110.