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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Molecular cloning, expression, and purification of SARS-CoV nsp13</title><author><name sortKey="Fan, Zheng" sort="Fan, Zheng" uniqKey="Fan Z" first="Zheng" last="Fan">Zheng Fan</name><affiliation><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff></affiliation></author><author><name sortKey="Peng, Kunpeng" sort="Peng, Kunpeng" uniqKey="Peng K" first="Kunpeng" last="Peng">Kunpeng Peng</name><affiliation><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff></affiliation></author><author><name sortKey="Tan, Xinyu" sort="Tan, Xinyu" uniqKey="Tan X" first="Xinyu" last="Tan">Xinyu Tan</name><affiliation><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff></affiliation></author><author><name sortKey="Yin, Bin" sort="Yin, Bin" uniqKey="Yin B" first="Bin" last="Yin">Bin Yin</name><affiliation><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff></affiliation></author><author><name sortKey="Dong, Xiuhua" sort="Dong, Xiuhua" uniqKey="Dong X" first="Xiuhua" last="Dong">Xiuhua Dong</name><affiliation><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff></affiliation></author><author><name sortKey="Qiu, Feichan" sort="Qiu, Feichan" uniqKey="Qiu F" first="Feichan" last="Qiu">Feichan Qiu</name><affiliation><nlm:aff id="aff2">Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China</nlm:aff></affiliation></author><author><name sortKey="Shen, Yan" sort="Shen, Yan" uniqKey="Shen Y" first="Yan" last="Shen">Yan Shen</name><affiliation><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff></affiliation></author><author><name sortKey="Wang, Heng" sort="Wang, Heng" uniqKey="Wang H" first="Heng" last="Wang">Heng Wang</name><affiliation><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff></affiliation></author><author><name sortKey="Yuan, Jiangang" sort="Yuan, Jiangang" uniqKey="Yuan J" first="Jiangang" last="Yuan">Jiangang Yuan</name><affiliation><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff></affiliation></author><author><name sortKey="Qiang, Boqin" sort="Qiang, Boqin" uniqKey="Qiang B" first="Boqin" last="Qiang">Boqin Qiang</name><affiliation><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff></affiliation></author><author><name sortKey="Peng, Xiaozhong" sort="Peng, Xiaozhong" uniqKey="Peng X" first="Xiaozhong" last="Peng">Xiaozhong Peng</name><affiliation><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">15866708</idno><idno type="pmc">7129550</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7129550</idno><idno type="RBID">PMC:7129550</idno><idno type="doi">10.1016/j.pep.2004.08.003</idno><date when="2005">2005</date><idno type="wicri:Area/Pmc/Corpus">001444</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">001444</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Molecular cloning, expression, and purification of SARS-CoV nsp13</title><author><name sortKey="Fan, Zheng" sort="Fan, Zheng" uniqKey="Fan Z" first="Zheng" last="Fan">Zheng Fan</name><affiliation><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff></affiliation></author><author><name sortKey="Peng, Kunpeng" sort="Peng, Kunpeng" uniqKey="Peng K" first="Kunpeng" last="Peng">Kunpeng Peng</name><affiliation><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff></affiliation></author><author><name sortKey="Tan, Xinyu" sort="Tan, Xinyu" uniqKey="Tan X" first="Xinyu" last="Tan">Xinyu Tan</name><affiliation><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff></affiliation></author><author><name sortKey="Yin, Bin" sort="Yin, Bin" uniqKey="Yin B" first="Bin" last="Yin">Bin Yin</name><affiliation><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff></affiliation></author><author><name sortKey="Dong, Xiuhua" sort="Dong, Xiuhua" uniqKey="Dong X" first="Xiuhua" last="Dong">Xiuhua Dong</name><affiliation><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff></affiliation></author><author><name sortKey="Qiu, Feichan" sort="Qiu, Feichan" uniqKey="Qiu F" first="Feichan" last="Qiu">Feichan Qiu</name><affiliation><nlm:aff id="aff2">Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China</nlm:aff></affiliation></author><author><name sortKey="Shen, Yan" sort="Shen, Yan" uniqKey="Shen Y" first="Yan" last="Shen">Yan Shen</name><affiliation><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff></affiliation></author><author><name sortKey="Wang, Heng" sort="Wang, Heng" uniqKey="Wang H" first="Heng" last="Wang">Heng Wang</name><affiliation><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff></affiliation></author><author><name sortKey="Yuan, Jiangang" sort="Yuan, Jiangang" uniqKey="Yuan J" first="Jiangang" last="Yuan">Jiangang Yuan</name><affiliation><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff></affiliation></author><author><name sortKey="Qiang, Boqin" sort="Qiang, Boqin" uniqKey="Qiang B" first="Boqin" last="Qiang">Boqin Qiang</name><affiliation><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff></affiliation></author><author><name sortKey="Peng, Xiaozhong" sort="Peng, Xiaozhong" uniqKey="Peng X" first="Xiaozhong" last="Peng">Xiaozhong Peng</name><affiliation><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff></affiliation></author></analytic><series><title level="j">Protein Expression and Purification</title><idno type="ISSN">1046-5928</idno><idno type="eISSN">1096-0279</idno><imprint><date when="2005">2005</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The SARS-nsp13 protein was identified as an mRNA cap1 methyltransferase. In this study, the nsp13 gene was cloned from the SARS-CoV PUMC02 strain viral RNA by RT-PCR, and inserted into the expression plasmid pET30a(+). The recombinant plasmid pET30a(+)-nsp13 was confirmed by restriction enzymes and sequencing analysis, and transformed into <italic>Escherichia coli</italic> BL21(DE3). The His-tag-fused protein was expressed by induction of 0.5 mM IPTG and purified by a single Ni<sup>2+</sup> affinity chromatography. The protein was validated by western blot and MS analysis. A large quantity of the nsp13 protein obtained with this method may be useful for further study of its structure and function.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Ksiazek, T G" uniqKey="Ksiazek T">T.G. Ksiazek</name></author><author><name sortKey="Erdman, D" uniqKey="Erdman D">D. Erdman</name></author><author><name sortKey="Goldsmith, C S" uniqKey="Goldsmith C">C.S. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Protein Expr Purif</journal-id><journal-id journal-id-type="iso-abbrev">Protein Expr. Purif</journal-id><journal-title-group><journal-title>Protein Expression and Purification</journal-title></journal-title-group><issn pub-type="ppub">1046-5928</issn><issn pub-type="epub">1096-0279</issn><publisher><publisher-name>Elsevier Inc.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">15866708</article-id><article-id pub-id-type="pmc">7129550</article-id><article-id pub-id-type="publisher-id">S1046-5928(04)00278-5</article-id><article-id pub-id-type="doi">10.1016/j.pep.2004.08.003</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Molecular cloning, expression, and purification of SARS-CoV nsp13</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Fan</surname><given-names>Zheng</given-names></name><xref rid="aff1" ref-type="aff">a</xref><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author"><name><surname>Peng</surname><given-names>Kunpeng</given-names></name><xref rid="aff1" ref-type="aff">a</xref><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author"><name><surname>Tan</surname><given-names>Xinyu</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Yin</surname><given-names>Bin</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Dong</surname><given-names>Xiuhua</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Qiu</surname><given-names>Feichan</given-names></name><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Shen</surname><given-names>Yan</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Heng</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Yuan</surname><given-names>Jiangang</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Qiang</surname><given-names>Boqin</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Peng</surname><given-names>Xiaozhong</given-names></name><email>pengxiaozhong@pumc.edu.cn</email><xref rid="aff1" ref-type="aff">a</xref><xref rid="cor1" ref-type="corresp">⁎</xref></contrib></contrib-group><aff id="aff1"><label>a</label>National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</aff><aff id="aff2"><label>b</label>Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China</aff><author-notes><corresp id="cor1"><label>⁎</label>Corresponding author. Fax: +86 10 65240529 <email>pengxiaozhong@pumc.edu.cn</email></corresp><fn id="fn1"><label>1</label><p>These two authors contributed equally to this work.</p></fn></author-notes><pub-date pub-type="pmc-release"><day>11</day><month>4</month><year>2005</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="ppub"><month>6</month><year>2005</year></pub-date><pub-date pub-type="epub"><day>11</day><month>4</month><year>2005</year></pub-date><volume>41</volume><issue>2</issue><fpage>235</fpage><lpage>240</lpage><history><date date-type="received"><day>28</day><month>4</month><year>2004</year></date><date date-type="rev-recd"><day>29</day><month>7</month><year>2004</year></date></history><permissions><copyright-statement>Copyright © 2005 Elsevier Inc. All rights reserved.</copyright-statement><copyright-year>2005</copyright-year><copyright-holder>Elsevier Inc.</copyright-holder><license><license-p>Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.</license-p></license></permissions><abstract><p>The SARS-nsp13 protein was identified as an mRNA cap1 methyltransferase. In this study, the nsp13 gene was cloned from the SARS-CoV PUMC02 strain viral RNA by RT-PCR, and inserted into the expression plasmid pET30a(+). The recombinant plasmid pET30a(+)-nsp13 was confirmed by restriction enzymes and sequencing analysis, and transformed into <italic>Escherichia coli</italic> BL21(DE3). The His-tag-fused protein was expressed by induction of 0.5 mM IPTG and purified by a single Ni<sup>2+</sup> affinity chromatography. The protein was validated by western blot and MS analysis. A large quantity of the nsp13 protein obtained with this method may be useful for further study of its structure and function.</p></abstract><kwd-group><title>Keywords</title><kwd>Severe acute respiratory syndrome (SARS)</kwd><kwd>nsp13 (SARS-CoV methyltransferase)</kwd><kwd>Molecular cloning</kwd><kwd>Expression</kwd><kwd>Purification</kwd><kwd>Mass spectrometric identification</kwd></kwd-group></article-meta></front><body><p>In the spring of 2003, a severe epidemic disease broke out in China and quickly spread to more than 30 countries. It led to over 8000 infected cases and as many as 774 deaths <xref rid="bib1" ref-type="bibr">[1]</xref>. This disease was designated as severe acute respiratory syndrome or SARS by the World Health Organization (WHO) and the pathogen was identified as a novel coronavirus, named SARS-associated coronavirus (SARS-CoV) <xref rid="bib2" ref-type="bibr">[2]</xref>, <xref rid="bib3" ref-type="bibr">[3]</xref>, <xref rid="bib4" ref-type="bibr">[4]</xref>. The SARS-CoV is a single-stranded plus-sense RNA virus with a genome of about 30 kb in length that has a 5′ cap structure and a 3′ polyadenylation tail. Upon infection of an appropriate host cell, the 5′-most open-reading frame (ORF) of the viral genome is translated into a large polyprotein that is cleaved by proteases to release several nonstructural proteins (nsps), including nsp2 (3C-like proteinase), nsp9 (RNA-dependent RNA polymerase, RdRp), nsp10 (NTPase/HEL), and so on <xref rid="bib5" ref-type="bibr">[5]</xref>, <xref rid="bib6" ref-type="bibr">[6]</xref>. Among these nsps, the nsp13 protein has been predicted as an mRNA cap 1 methyltransferase by 3D jury system <xref rid="bib7" ref-type="bibr">[7]</xref>.</p><p>Methylated 5′-terminal cap structures have been described in most eukaryotic and many viral mRNAs. The methylated cap structure is essential for efficient initiation of translation and mRNA stability <xref rid="bib8" ref-type="bibr">[8]</xref>. In all cap structures, including the “minimal” cap 0 (m7G(5′)ppp(5′)N), an <italic>N</italic>7-methylguanosine (m7G) is attached through a 5′–5′ triphosphate bridge to the penultimate nucleoside. In some molecules, additional 2′-<italic>O</italic>-ribose methylations are found at the penultimate and the antepenultimate nucleosides, forming the cap 1 (m7G(5′)ppp(5′)Nm) and cap 2 (m7G(5′)ppp(5′)NmpNm) structures, respectively <xref rid="bib9" ref-type="bibr">[9]</xref>, <xref rid="bib10" ref-type="bibr">[10]</xref>. The enzyme that catalyzes the cap 1 structure is a (nucleoside-2′-<italic>O</italic>-)-methyltransferase (2′-<italic>O</italic>-MTase), which usually utilizes <italic>S</italic>-adenosyl-<sc>l</sc>-methionine (AdoMet) as the methyl donor. The SARS-nsp13 protein belongs to the ancient family of AdoMet-dependent ribose 2′-<italic>O</italic>-MTase. The enzymatic role of the protein was confirmed by the presence of the conserved tetrad of residues K–D–K–E essential for mRNA cap 1 formation <xref rid="bib7" ref-type="bibr">[7]</xref>.</p><p>Viruses adopt different capping strategies based on their replication cycles and hosts, but most of them have the same cap structure as cellular mRNA. Viruses with cytoplamic replication cycle have to synthesize their 5′-terminal cap structures with their own methyltransferases. The order of methyl transfer reactions is variable, in some viruses the cap 0 structure is necessary for cap 1 methylation <xref rid="bib10" ref-type="bibr">[10]</xref>, and in other viruses the cap 1 is a preferred substrate for the cap 0 MTase <xref rid="bib11" ref-type="bibr">[11]</xref>, <xref rid="bib12" ref-type="bibr">[12]</xref>. So viral cap-forming enzymes (cap 0 and/or cap 1) are potential targets for antiviral drugs that would interfere with capping of pathogen mRNAs but spare the host capping enzymes. Exploring the structure and function of the SARS-CoV nsp13 protein will be extremely useful for the development of anti-SARS agents.</p><p>In this study, we cloned the nsp13 gene by RT-PCR from SARS-CoV PUMC02 strain genomic RNA, and constructed a recombinant expression vector pET30a(+)-nsp13. The His-tag-fused SARS-nsp13 protein was then expressed in <italic>Escherichia coli</italic>, purified through a single step affinity chromatography, and finally identified by both western blot and mass spectrometry. A large quantity of the nsp13 protein can be obtained with this method.</p><sec><title>Materials and methods</title><sec><title>Reagents</title><p>Restriction enzymes were purchased from New England Biolabs (Berverly, MA). The expand reverse transcriptase and modified bovine trypsin (sequencing grade) were obtained from Roche (Mannheim, Germany). Pfu DNA polymerase and T4 DNA ligase were from Shanghai Sangon Co (Shanghai, China). Trizol reagent was from Invitrogen (Carlsbad, CA). The plasmid extraction kit and DNA agarose gel cleanup kit were obtained from BioDev (Beijing, China). Ni<sup>2+</sup> -NTA resin was purchased from Qiagen (Valencia, CA). Nitrocellulose membrane was purchased from Amersham Life Science (Newington, NH), and His-tag monoclonal antibody was from Novagen (Darmstadt, Germany).</p></sec><sec><title>Construction of nsp13 gene in the expression vector pET30a(+)</title><p>All cloning techniques including PCR, restriction digestion, ligation, and <italic>E. coli</italic> transformation were performed as previously published <xref rid="bib13" ref-type="bibr">[13]</xref>.</p><p>SARS-CoV (PUMC02 strain) RNA was extracted with Trizol reagent according to the manufacturer’s instruction. The reverse transcription was performed by the expand reverse transcriptase with the primer TTGTT AACAA GAATA TCACT TGAAA CCAC, which is proximately downstream of the nsp13 gene. The SARS nsp13 cDNA was subsequently amplified by PCR, using the following primers, nsp13 forward, 5′-CG<underline>GGA</underline><underline>TCC</underline>GC AAGTC AAGCG TGGCA AC-3′, and nsp13 reverse 5′-CCG<underline>CT</underline><underline>CGAG</underline>T TAGTT GTTAA CAAGA ATATC AC-3′, the sequences underlined are the recognition sites of the restriction enzymes <italic>Bam</italic>HI and <italic>Xho</italic>I. After digestion with <italic>Bam</italic>HI and <italic>XhoI</italic>, the PCR product was purified with gel cleanup kit and inserted between the <italic>Bam</italic>HI and <italic>Xho</italic>I sites of the vector pET30a(+). The recombinant vector pET30a(+)-nsp13 was identified by restriction digestion and the SARS-nsp13 insert was verified by sequencing. <italic>E. coli</italic> DH5α was used for amplification of the recombinant plasmid and <italic>E. coli</italic> BL21(DE3) was transformed for induced expression of the His-tagged SARS-nsp13 protein.</p></sec><sec><title>Expression and purification of SARS-nsp13 protein</title><p>A single colony of <italic>E. coli</italic> BL21(DE3) cells transformed with the recombinant expression plasmid pET30a(+)-nsp13 was inoculated in a tube containing 2 ml of LB medium with 50 μg/ml kanamycin, and cultured overnight at 37 °C in a shaking incubator. The culture was transferred to a 1–L flask containing 200 ml of the same medium. The flask was shaken at 37 °C until the OD<sub>600</sub> of the culture was about 0.6–0.8. The expression of SARS-nsp13 protein was induced by the addition of 0.5 mM of isopropyl β-<sc>d</sc>-thiogalactoside (IPTG). After induction for 5 h at 25 °C, the cells were harvested by centrifugation at 4000<italic>g</italic>, 4 °C for 30 min. The supernatant was discarded and the cell pellet was washed, frozen, resuspended in PBS (pH 8.0), and then disrupted by sonication. The lysed cells were centrifuged at 12,000<italic>g</italic>, 4 °C for 30 min. The supernatant was filtered through a 0.45 μm syringe filter. Ni<sup>2+</sup> chelating resin (200 μl) was equilibrated with 5 ml of sterile deionized water, 2 ml of 50 mM NiSO<sub>4</sub>, and 5 ml of PBS. The supernatant was passed through the column, followed by washing the column with 5 ml of PBS and 5 ml of Wash Buffer (20 mM imidazole in PBS), respectively. The protein of interest was then eluted with 1–2 ml of elution buffer (200 mM imidazole in PBS). The eluted fractions were analyzed by 12% SDS–PAGE.</p></sec><sec><title>Immunoblot analysis of SARS-nsp13 protein by His-tag monoclonal antibody</title><p>The protein sample was resolved on 12% SDS–PAGE gel and transferred onto nitrocellulose membrane using the Bio-Rad trans-blot apparatus. The membrane was incubated for 2 h at room temperature in Buffer I (5% skim milk, 0.02% Tween-20), His-tag monoclonal antibody was then added at a dilution of 1:1000, and the membrane was incubated at 4 °C overnight. Three washes were followed by addition of alkaline phosphatase-conjugated horse anti-mouse IgG at a dilution of 1:1000 in Buffer II (5% skim milk, 150 mM NaCl, 50 mM Tris–Cl, and pH 7.5), the membrane was incubated for 2 h at room temperature. After three washes with PBS, the membrane was immerged into Buffer III (100 mM NaCl, 5 mM MgCl<sub>2</sub>, and 100 mM Tris–Cl, pH 9.5) containing alkaline phosphatase substrate of 5-bromo-4-chloro-3-indoxyl phosphate (BCIP) and nitroblue tetrazolium (NBT). The blot was allowed to develop and the reaction was stopped by washing the membrane in distilled water.</p></sec><sec><title>Mass spectrometric identification of SARS-nsp13 protein</title><p>Excised gel band of interest was in-gel digested with modified bovine trypsin <xref rid="bib14" ref-type="bibr">[14]</xref>. Excess Coomassie brilliant blue stain was removed by washing twice with 1 ml of 100 mM NH<sub>4</sub>HCO<sub>3</sub>, 50% acetonitrile for 1 h at room temperature. Then clear gel pieces were vacuum dried completely using a SpeedVac concentrator (Savant). The dried gel pieces were re-hydrated by adding 10 μl of digestion buffer (50 mM NH<sub>4</sub>HCO<sub>3</sub>) containing 0.5 μg of trypsin. After allowing 30 min for gel pieces to completely absorb the solution, an additional 200 μl of digestion buffer without trypsin was added to fully immerse the gel pieces. Digestion continued for about 16 h at 37 °C. The digestion buffer, now containing extracted peptides, was carefully removed and transferred into a clean microfuge tube. The gel pieces were re-extracted twice by adding 100 μl of 0.1% TFA in 50% acetonitrile and incubating for 30 min at room temperature. The pooled extracts were vacuum concentrated to reduce the volume to less than 10 μl. A volume of 0.5 μl of matrix solution (α-cyano-4-hydroxycinnamic acid saturated in 50% v/v acetonitrile/0.5% v/v TFA) was mixed with the same volume of the extract, then applied onto stainless steel target. Mass spectra were recorded in positive ion reflection mode of a matrix assisted laser desorption ionization-time of flight (MALDI-ToF) Voyager DE PRO (Applied Biosystems). Peptide masses obtained were searched against NCBInr database without any species limit using the Mascot search engine available online (<ext-link ext-link-type="uri" xlink:href="http://www.matrixscience.com">www.matrixscience.com</ext-link>).</p></sec></sec><sec><title>Results and discussion</title><sec><title>Construction of the expression vector pET30a(+)-nsp13</title><p>The SARS-nsp13 gene was amplified by RT-PCR (<xref rid="fig1" ref-type="fig">Fig. 1</xref>A), the amplified product was about 900 bp, which was accordant to the theoretical length of nsp13 gene. The recombinant plasmid was digested by the restriction enzymes <italic>Bam</italic>HI and <italic>Xho</italic>I. Agarose gel electrophoresis of the digest revealed a DNA band at about 900 bp (<xref rid="fig1" ref-type="fig">Fig. 1</xref>B). The inserted fragment was verified by sequencing, the result was shown in <xref rid="fig2" ref-type="fig">Fig. 2</xref>, which was identical to the published SARS-nsp13 gene sequence in NCBI and in the correct reading frame with the His-tag in the vector.<fig id="fig1"><label>Fig. 1</label><caption><p>PCR product of SARS-CoV PUMC02 strain-nsp13 gene (A) and the recombinant expression vector pET30a(+)-nsp13 digestion with restriction enzymes <italic>BamHI</italic> and <italic>XhoI</italic> (B).</p></caption><graphic xlink:href="gr1"></graphic></fig><fig id="fig2"><label>Fig. 2</label><caption><p>Sequencing result of the cloned SARS-CoV PUMC02 strain-nsp13 gene and the translated amino acid sequence. The bold and italic letters indicate the conserved tetrad of residues K–D–K–E.</p></caption><graphic xlink:href="gr2"></graphic></fig></p></sec><sec><title>Expression and purification of SARS-nsp13 protein</title><p><italic>Escherichia coli</italic> BL21(DE3) was transformed with the recombinant expression vector pET30a(+)-nsp13, and SARS-nsp13 protein was abundantly expressed after induction by IPTG . As shown in <xref rid="fig3" ref-type="fig">Fig. 3</xref>A, some of the target protein existed in the pellet. Lowering culture temperature and reducing the concentration of IPTG did not change the solubility (data is not shown here). The soluble fraction was loaded onto the Ni<sup>2+</sup> -NTA affinity column, and the homogeneous protein was successfully purified with elution buffer after washing thoroughly. The purified protein was further analyzed with His-tag monoclonal antibody, and a specific blot band was detected at the corresponding position (<xref rid="fig3" ref-type="fig">Fig. 3</xref>B). The predicted molecular mass of the SARS-nsp13 protein was 33.4 kDa, and there were 30 additional amino acids for the His-tag in the N-terminal of the expressed fusion protein. These extra amino acids increased molecular mass of the expressed target protein by about 3.3 kDa. Apparent molecular mass of the purified protein was about 37 kDa, which was expected. The yield from 200 ml of bacterial culture was about 1.3 mg of purified SARS-nsp13 protein after a single Ni<sup>2+</sup> affinity chromatographic step. The recovery of SARS-nsp13 protein was about 26% (<xref rid="tbl1" ref-type="table">Table 1</xref>).<fig id="fig3"><label>Fig. 3</label><caption><p>(A) 12% SDS–PAGE analysis of the expression and purification of SARS-nsp13 protein. 1, uninduced; 2, induced; 3, pellet; 4, supernatant; 5, flow-through; 6, purified nsp13 protein by Ni<sup>2+</sup>-NTA affinity column; and M, Low molecular weight protein marker. (B) Western blot analysis of the SARS-nsp13 protein by His-tag monoclonal antibody.</p></caption><graphic xlink:href="gr3"></graphic></fig><table-wrap position="float" id="tbl1"><label>Table 1</label><caption><p>Purification scheme of SARS-nsp13 protein expressed in <italic>E. coli</italic> BL21(DE3) with pET30a(+)-nsp13 (200 ml culture)</p></caption><table frame="hsides" rules="groups"><thead><tr><th>Step</th><th>Total protein (mg)</th><th>nsp13 protein (mg)</th><th>Recovery nsp13 (%)</th></tr></thead><tbody><tr><td>Extraction</td><td align="char">67.8</td><td>4.9</td><td align="char">100</td></tr><tr><td>Ni<sup>2+</sup>-affinity column</td><td align="char">1.3</td><td>1.3</td><td align="char">26</td></tr></tbody></table></table-wrap></p></sec><sec><title>MALDI-ToF MS identification of SARS-nsp13 protein</title><p>The MALDI-ToF mass spectrum of tryptic digest of the gel band was shown in <xref rid="fig4" ref-type="fig">Fig. 4</xref>. The achieved peptide masses were searched against NCBInr database without any species limit and with peptide mass tolerance of ±0.1 Da using the Mascot search engine. The first candidate protein, with a score of 94, was SARS nsp16 protein (GI:30133975), a putative ribose 2′-<italic>O</italic>-methyltransferase (the sequence was identical to that of the detected nsp13 protein here), and was the only one having a score greater than 75. The second candidate protein had a score of 67, which was lower than the significant score of 75. Seven peptide fragments were matched with tryptic peptides of nsp13 protein, and the sequence coverage was 26%. The covered portions in nsp13 protein were underlined in <xref rid="fig5" ref-type="fig">Fig. 5</xref>. The results above indubitably determined the identity of the recombinant His-tagged SARS-nsp13 protein.<fig id="fig4"><label>Fig. 4</label><caption><p>MALDI-ToF mass spectrum of the tryptic digest of His-tagged SARS-nsp13 protein.</p></caption><graphic xlink:href="gr4"></graphic></fig><fig id="fig5"><label>Fig. 5</label><caption><p>Matched peptide fragments in SARS-nsp13 protein. The matched portions are shown in bold and underlined.</p></caption><graphic xlink:href="gr5"></graphic></fig></p><p>In conclusion, we have succeeded in the molecular cloning of SARS-nsp13, and have transformed the recombinant plasmid pET30a(+)-nsp13 into <italic>E. coli</italic> BL21(DE3). The purified His-tagged nsp13 protein has been identified by both western blot and MALDI-ToF mass spectrometry. The expression and purification procedures in this study have provided a simple and efficient method to obtain pure nsp13 protein of SARS-CoV in large quantities. 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