NT-HRV3CP: An optimized construct of human rhinovirus 14 3C protease for high-yield expression and fast affinity-tag cleavage
[Display omitted] •NT* fusion of human rhinovirus 14 3C protease for high-yield expression.•Stable protease for tag removal at low temperature in minutes.•Alternative to tag cleavage with TEV protease. The human rhinovirus 14 3C protease (HRV3C protease), in fusion with glutathione S-transferase als...
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| Veröffentlicht in: | Journal of biotechnology 2021-01, Vol.325, p.145-151 |
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| container_title | Journal of biotechnology |
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| creator | Abdelkader, Elwy H. Otting, Gottfried |
| description | [Display omitted]
•NT* fusion of human rhinovirus 14 3C protease for high-yield expression.•Stable protease for tag removal at low temperature in minutes.•Alternative to tag cleavage with TEV protease.
The human rhinovirus 14 3C protease (HRV3C protease), in fusion with glutathione S-transferase also referred to as PreScission™ protease, is a cysteine protease of particular interest for affinity tag removal from fusion proteins due to its stringent recognition sequence specificity (LEVLFQ/GX) and superior activity at low temperature. Here we report the expression, purification and use of a fusion construct of HRV3C protease, NT*-HRV3CP, that affords high expression yield in E. coli (over 300 mg/L cell culture), facile single-step purification, high solubility (>10 mg/mL) and excellent storage properties. NT*-HRV3CP cleaves affinity tags at 4 °C in minutes, making it an attractive tool for the production of recombinant proteins for biotechnological, industrial and pharmaceutical applications. |
| doi_str_mv | 10.1016/j.jbiotec.2020.11.005 |
| format | Article |
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•NT* fusion of human rhinovirus 14 3C protease for high-yield expression.•Stable protease for tag removal at low temperature in minutes.•Alternative to tag cleavage with TEV protease.
The human rhinovirus 14 3C protease (HRV3C protease), in fusion with glutathione S-transferase also referred to as PreScission™ protease, is a cysteine protease of particular interest for affinity tag removal from fusion proteins due to its stringent recognition sequence specificity (LEVLFQ/GX) and superior activity at low temperature. Here we report the expression, purification and use of a fusion construct of HRV3C protease, NT*-HRV3CP, that affords high expression yield in E. coli (over 300 mg/L cell culture), facile single-step purification, high solubility (>10 mg/mL) and excellent storage properties. NT*-HRV3CP cleaves affinity tags at 4 °C in minutes, making it an attractive tool for the production of recombinant proteins for biotechnological, industrial and pharmaceutical applications.</description><identifier>ISSN: 0168-1656</identifier><identifier>EISSN: 1873-4863</identifier><identifier>DOI: 10.1016/j.jbiotec.2020.11.005</identifier><identifier>PMID: 33166527</identifier><language>eng</language><publisher>Netherlands: Elsevier B.V</publisher><subject>3C Viral Proteases ; Chromatography, Affinity ; Cysteine Endopeptidases - genetics ; Enterovirus ; Escherichia coli - genetics ; Fusion tag cleavage ; Human rhinovirus 14 3C protease ; Humans ; NT fusion protein ; Peptide Hydrolases ; PreScission protease ; Recombinant Fusion Proteins - genetics ; Rhinovirus - genetics</subject><ispartof>Journal of biotechnology, 2021-01, Vol.325, p.145-151</ispartof><rights>2020 Elsevier B.V.</rights><rights>Copyright © 2020 Elsevier B.V. All rights reserved.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c412t-c7141018fda4706df8ad12e7f8be443c90fd6aa7152fff4f429c96d88a8dc2c03</citedby><cites>FETCH-LOGICAL-c412t-c7141018fda4706df8ad12e7f8be443c90fd6aa7152fff4f429c96d88a8dc2c03</cites><orcidid>0000-0002-0563-0146 ; 0000-0002-5388-3949</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.jbiotec.2020.11.005$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/33166527$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Abdelkader, Elwy H.</creatorcontrib><creatorcontrib>Otting, Gottfried</creatorcontrib><title>NT-HRV3CP: An optimized construct of human rhinovirus 14 3C protease for high-yield expression and fast affinity-tag cleavage</title><title>Journal of biotechnology</title><addtitle>J Biotechnol</addtitle><description>[Display omitted]
•NT* fusion of human rhinovirus 14 3C protease for high-yield expression.•Stable protease for tag removal at low temperature in minutes.•Alternative to tag cleavage with TEV protease.
The human rhinovirus 14 3C protease (HRV3C protease), in fusion with glutathione S-transferase also referred to as PreScission™ protease, is a cysteine protease of particular interest for affinity tag removal from fusion proteins due to its stringent recognition sequence specificity (LEVLFQ/GX) and superior activity at low temperature. Here we report the expression, purification and use of a fusion construct of HRV3C protease, NT*-HRV3CP, that affords high expression yield in E. coli (over 300 mg/L cell culture), facile single-step purification, high solubility (>10 mg/mL) and excellent storage properties. NT*-HRV3CP cleaves affinity tags at 4 °C in minutes, making it an attractive tool for the production of recombinant proteins for biotechnological, industrial and pharmaceutical applications.</description><subject>3C Viral Proteases</subject><subject>Chromatography, Affinity</subject><subject>Cysteine Endopeptidases - genetics</subject><subject>Enterovirus</subject><subject>Escherichia coli - genetics</subject><subject>Fusion tag cleavage</subject><subject>Human rhinovirus 14 3C protease</subject><subject>Humans</subject><subject>NT fusion protein</subject><subject>Peptide Hydrolases</subject><subject>PreScission protease</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Rhinovirus - genetics</subject><issn>0168-1656</issn><issn>1873-4863</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkFGLEzEUhYMobq3-BCWPvkxNMplMxhdZyuoKi4qsvoY0uWlTZpKaZIoV_O9mafXVpwuXc-6550PoJSUrSqh4s1_tNz4WMCtGWN3RFSHdI7Sgsm8bLkX7GC2qTjZUdOIKPct5TwjhQ0efoqu2pUJ0rF-g35_um9uv39v1l7f4OuB4KH7yv8BiE0MuaTYFR4d386QDTjsf4tGnOWPKcbvGh1Qf0Bmwiwnv_HbXnDyMFsPPQ4KcfQxYB4udzgVr53zw5dQUvcVmBH3UW3iOnjg9ZnhxmUv07f3N_fq2ufv84eP6-q4xnLLSmJ7yWlo6q3lPhHVSW8qgd3IDnLdmIM4KrXvaMeccd5wNZhBWSi2tYYa0S_T6fLd-_GOGXNTks4Fx1AHinBXj3dB2fV-xLVF3lpoUc07g1CH5SaeTokQ9kFd7dSGvHsgrSlUlX32vLhHzZgL7z_UXdRW8OwugFj16SCobD8GA9QlMUTb6_0T8AWndmCA</recordid><startdate>20210110</startdate><enddate>20210110</enddate><creator>Abdelkader, Elwy H.</creator><creator>Otting, Gottfried</creator><general>Elsevier B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0002-0563-0146</orcidid><orcidid>https://orcid.org/0000-0002-5388-3949</orcidid></search><sort><creationdate>20210110</creationdate><title>NT-HRV3CP: An optimized construct of human rhinovirus 14 3C protease for high-yield expression and fast affinity-tag cleavage</title><author>Abdelkader, Elwy H. ; Otting, Gottfried</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c412t-c7141018fda4706df8ad12e7f8be443c90fd6aa7152fff4f429c96d88a8dc2c03</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>3C Viral Proteases</topic><topic>Chromatography, Affinity</topic><topic>Cysteine Endopeptidases - genetics</topic><topic>Enterovirus</topic><topic>Escherichia coli - genetics</topic><topic>Fusion tag cleavage</topic><topic>Human rhinovirus 14 3C protease</topic><topic>Humans</topic><topic>NT fusion protein</topic><topic>Peptide Hydrolases</topic><topic>PreScission protease</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Rhinovirus - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Abdelkader, Elwy H.</creatorcontrib><creatorcontrib>Otting, Gottfried</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Abdelkader, Elwy H.</au><au>Otting, Gottfried</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>NT-HRV3CP: An optimized construct of human rhinovirus 14 3C protease for high-yield expression and fast affinity-tag cleavage</atitle><jtitle>Journal of biotechnology</jtitle><addtitle>J Biotechnol</addtitle><date>2021-01-10</date><risdate>2021</risdate><volume>325</volume><spage>145</spage><epage>151</epage><pages>145-151</pages><issn>0168-1656</issn><eissn>1873-4863</eissn><abstract>[Display omitted]
•NT* fusion of human rhinovirus 14 3C protease for high-yield expression.•Stable protease for tag removal at low temperature in minutes.•Alternative to tag cleavage with TEV protease.
The human rhinovirus 14 3C protease (HRV3C protease), in fusion with glutathione S-transferase also referred to as PreScission™ protease, is a cysteine protease of particular interest for affinity tag removal from fusion proteins due to its stringent recognition sequence specificity (LEVLFQ/GX) and superior activity at low temperature. Here we report the expression, purification and use of a fusion construct of HRV3C protease, NT*-HRV3CP, that affords high expression yield in E. coli (over 300 mg/L cell culture), facile single-step purification, high solubility (>10 mg/mL) and excellent storage properties. NT*-HRV3CP cleaves affinity tags at 4 °C in minutes, making it an attractive tool for the production of recombinant proteins for biotechnological, industrial and pharmaceutical applications.</abstract><cop>Netherlands</cop><pub>Elsevier B.V</pub><pmid>33166527</pmid><doi>10.1016/j.jbiotec.2020.11.005</doi><tpages>7</tpages><orcidid>https://orcid.org/0000-0002-0563-0146</orcidid><orcidid>https://orcid.org/0000-0002-5388-3949</orcidid><oa>free_for_read</oa></addata></record> |
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| ispartof | Journal of biotechnology, 2021-01, Vol.325, p.145-151 |
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| subjects | 3C Viral Proteases Chromatography, Affinity Cysteine Endopeptidases - genetics Enterovirus Escherichia coli - genetics Fusion tag cleavage Human rhinovirus 14 3C protease Humans NT fusion protein Peptide Hydrolases PreScission protease Recombinant Fusion Proteins - genetics Rhinovirus - genetics |
| title | NT-HRV3CP: An optimized construct of human rhinovirus 14 3C protease for high-yield expression and fast affinity-tag cleavage |
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