A novel self-cleavable tag Zbasic–∆I-CM and its application in the soluble expression of recombinant human interleukin-15 in Escherichia coli
Soluble expression of recombinant therapeutic proteins in Escherichia coli ( E. coli ) has been a challenging task in biopharmaceutical development. In this study, a novel self-cleavable tag Zbasic–intein has been constructed for the soluble expression and purification of a recombinant cytokine, hum...
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| Veröffentlicht in: | Applied microbiology and biotechnology 2017-02, Vol.101 (3), p.1133-1142 |
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| creator | Shi, Siwei Chen, Huanhuan Jiang, Hua Xie, Yueqing Zhang, Lei Li, Ninghuan Zhu, Chencen Chen, Junsheng Luo, Han Wang, Jiaxian Feng, Lei Lu, Huili Zhu, Jianwei |
| description | Soluble expression of recombinant therapeutic proteins in
Escherichia coli
(
E. coli
) has been a challenging task in biopharmaceutical development. In this study, a novel self-cleavable tag Zbasic–intein has been constructed for the soluble expression and purification of a recombinant cytokine, human interleukin-15 (IL-15). We screened several solubilizing tags fused with the self-cleavable
Mycobacterium tuberculosis
recA mini-intein ∆I-CM and demonstrated that Zbasic tag can significantly improve the solubility of the product with correspondent to the intein activity. The fusion protein “Zbasic–∆I-CM–IL-15” was expressed with high solubility and easily enriched by the cost-effective cation-exchange chromatography. The self-cleavage of the fusion tag Zbasic–∆I-CM was then induced by a pH shift, with an activation energy of 7.48 kcal/mol. The mature IL-15 with natural N-terminus was released and further purified by hydrophobic interaction and anion-exchange chromatography. High-resolution reverse-phase high-performance liquid chromatography and mass spectrometry analysis confirmed that the product was of high purity and correct mass. With a CTLL-2 cell proliferation-based assay, the EC
50
was evaluated to be of about 0.126 ng/mL, similar to the product in clinical trials. By avoiding the time-consuming denaturing-refolding steps in previously reported processes, the current method is efficient and cost-effective. The novel tag Zbasic–∆I-CM can be potentially applied to large-scale manufacturing of recombinant human cytokines as well as other mammalian-sourced proteins in
E. coli
. |
| doi_str_mv | 10.1007/s00253-016-7848-2 |
| format | Article |
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Escherichia coli
(
E. coli
) has been a challenging task in biopharmaceutical development. In this study, a novel self-cleavable tag Zbasic–intein has been constructed for the soluble expression and purification of a recombinant cytokine, human interleukin-15 (IL-15). We screened several solubilizing tags fused with the self-cleavable
Mycobacterium tuberculosis
recA mini-intein ∆I-CM and demonstrated that Zbasic tag can significantly improve the solubility of the product with correspondent to the intein activity. The fusion protein “Zbasic–∆I-CM–IL-15” was expressed with high solubility and easily enriched by the cost-effective cation-exchange chromatography. The self-cleavage of the fusion tag Zbasic–∆I-CM was then induced by a pH shift, with an activation energy of 7.48 kcal/mol. The mature IL-15 with natural N-terminus was released and further purified by hydrophobic interaction and anion-exchange chromatography. High-resolution reverse-phase high-performance liquid chromatography and mass spectrometry analysis confirmed that the product was of high purity and correct mass. With a CTLL-2 cell proliferation-based assay, the EC
50
was evaluated to be of about 0.126 ng/mL, similar to the product in clinical trials. By avoiding the time-consuming denaturing-refolding steps in previously reported processes, the current method is efficient and cost-effective. The novel tag Zbasic–∆I-CM can be potentially applied to large-scale manufacturing of recombinant human cytokines as well as other mammalian-sourced proteins in
E. coli
.</description><identifier>ISSN: 0175-7598</identifier><identifier>EISSN: 1432-0614</identifier><identifier>DOI: 10.1007/s00253-016-7848-2</identifier><identifier>PMID: 27683210</identifier><language>eng</language><publisher>Berlin/Heidelberg: Springer Berlin Heidelberg</publisher><subject>Analysis ; Biomedical and Life Sciences ; Biopharmaceutics - methods ; Biotechnologically Relevant Enzymes and Proteins ; Biotechnology ; Chromatography ; Chromatography, Liquid ; Clinical trials ; Cytokines ; E coli ; Enzymes ; Escherichia coli - genetics ; Escherichia coli - metabolism ; Gene expression ; Genetic recombination ; Humans ; Hydrogen-Ion Concentration ; Inteins ; Interleukin-15 - chemistry ; Interleukin-15 - genetics ; Interleukin-15 - isolation & purification ; Life Sciences ; Liquid chromatography ; Mass Spectrometry ; Microbial Genetics and Genomics ; Microbiology ; Mycobacterium tuberculosis - genetics ; Pharmaceutical sciences ; Plasmids ; Proteins ; Recombinant Fusion Proteins - chemistry ; Recombinant Fusion Proteins - economics ; Recombinant Fusion Proteins - isolation & purification ; Solubility ; Studies</subject><ispartof>Applied microbiology and biotechnology, 2017-02, Vol.101 (3), p.1133-1142</ispartof><rights>Springer-Verlag Berlin Heidelberg 2016</rights><rights>Applied Microbiology and Biotechnology is a copyright of Springer, 2017.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c3242-18e39fc5c2ca69f04b2a67252cfc3d50a13edf2da0a4c6efcf914de2ed1463403</citedby><cites>FETCH-LOGICAL-c3242-18e39fc5c2ca69f04b2a67252cfc3d50a13edf2da0a4c6efcf914de2ed1463403</cites><orcidid>0000-0002-8748-7899</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00253-016-7848-2$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00253-016-7848-2$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>315,781,785,27928,27929,41492,42561,51323</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27683210$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Shi, Siwei</creatorcontrib><creatorcontrib>Chen, Huanhuan</creatorcontrib><creatorcontrib>Jiang, Hua</creatorcontrib><creatorcontrib>Xie, Yueqing</creatorcontrib><creatorcontrib>Zhang, Lei</creatorcontrib><creatorcontrib>Li, Ninghuan</creatorcontrib><creatorcontrib>Zhu, Chencen</creatorcontrib><creatorcontrib>Chen, Junsheng</creatorcontrib><creatorcontrib>Luo, Han</creatorcontrib><creatorcontrib>Wang, Jiaxian</creatorcontrib><creatorcontrib>Feng, Lei</creatorcontrib><creatorcontrib>Lu, Huili</creatorcontrib><creatorcontrib>Zhu, Jianwei</creatorcontrib><title>A novel self-cleavable tag Zbasic–∆I-CM and its application in the soluble expression of recombinant human interleukin-15 in Escherichia coli</title><title>Applied microbiology and biotechnology</title><addtitle>Appl Microbiol Biotechnol</addtitle><addtitle>Appl Microbiol Biotechnol</addtitle><description>Soluble expression of recombinant therapeutic proteins in
Escherichia coli
(
E. coli
) has been a challenging task in biopharmaceutical development. In this study, a novel self-cleavable tag Zbasic–intein has been constructed for the soluble expression and purification of a recombinant cytokine, human interleukin-15 (IL-15). We screened several solubilizing tags fused with the self-cleavable
Mycobacterium tuberculosis
recA mini-intein ∆I-CM and demonstrated that Zbasic tag can significantly improve the solubility of the product with correspondent to the intein activity. The fusion protein “Zbasic–∆I-CM–IL-15” was expressed with high solubility and easily enriched by the cost-effective cation-exchange chromatography. The self-cleavage of the fusion tag Zbasic–∆I-CM was then induced by a pH shift, with an activation energy of 7.48 kcal/mol. The mature IL-15 with natural N-terminus was released and further purified by hydrophobic interaction and anion-exchange chromatography. High-resolution reverse-phase high-performance liquid chromatography and mass spectrometry analysis confirmed that the product was of high purity and correct mass. With a CTLL-2 cell proliferation-based assay, the EC
50
was evaluated to be of about 0.126 ng/mL, similar to the product in clinical trials. By avoiding the time-consuming denaturing-refolding steps in previously reported processes, the current method is efficient and cost-effective. The novel tag Zbasic–∆I-CM can be potentially applied to large-scale manufacturing of recombinant human cytokines as well as other mammalian-sourced proteins in
E. coli
.</description><subject>Analysis</subject><subject>Biomedical and Life Sciences</subject><subject>Biopharmaceutics - methods</subject><subject>Biotechnologically Relevant Enzymes and Proteins</subject><subject>Biotechnology</subject><subject>Chromatography</subject><subject>Chromatography, Liquid</subject><subject>Clinical trials</subject><subject>Cytokines</subject><subject>E coli</subject><subject>Enzymes</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - metabolism</subject><subject>Gene expression</subject><subject>Genetic recombination</subject><subject>Humans</subject><subject>Hydrogen-Ion Concentration</subject><subject>Inteins</subject><subject>Interleukin-15 - chemistry</subject><subject>Interleukin-15 - genetics</subject><subject>Interleukin-15 - isolation & purification</subject><subject>Life Sciences</subject><subject>Liquid chromatography</subject><subject>Mass Spectrometry</subject><subject>Microbial Genetics and Genomics</subject><subject>Microbiology</subject><subject>Mycobacterium tuberculosis - genetics</subject><subject>Pharmaceutical sciences</subject><subject>Plasmids</subject><subject>Proteins</subject><subject>Recombinant Fusion Proteins - chemistry</subject><subject>Recombinant Fusion Proteins - economics</subject><subject>Recombinant Fusion Proteins - isolation & purification</subject><subject>Solubility</subject><subject>Studies</subject><issn>0175-7598</issn><issn>1432-0614</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2017</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNp1kT1v1TAUhi0EopfCD2BBlli6mPorTjJWVy1UKuoCC4vlOMe9Lo4d7KSCrVPFiviH_SUkugUhJCYP53nfY50HoZeMvmGU1seFUl4JQpkidSMbwh-hDZOCE6qYfIw2lNUVqau2OUDPSrmmlPFGqafogNeqEZzRDfpxgmO6gYALBEdsAHNjugB4Mlf4U2eKt_e3P--_352T7XtsYo_9VLAZx-CtmXyK2Ec87QCXFOY1B1_HDKWsk-RwBpuGzkcTJ7ybB7PiE-QA82cfCavW9GmxO8je7rzBNgX_HD1xJhR48fAeoo9npx-278jF5dvz7ckFsYJLTlgDonW2stwa1ToqO25UzStunRV9RQ0T0DveG2qkVeCsa5nsgUPPpBKSikN0tO8dc_oyQ5n04IuFEEyENBfNmqqtRcMZX9DX_6DXac5x-d1CqYrLdrnmQrE9ZXMqJYPTY_aDyd80o3r1pfe-9OJLr770mnn10Dx3A_R_Er8FLQDfA2UZxSvIf63-b-svM0ejHg</recordid><startdate>20170201</startdate><enddate>20170201</enddate><creator>Shi, Siwei</creator><creator>Chen, Huanhuan</creator><creator>Jiang, Hua</creator><creator>Xie, Yueqing</creator><creator>Zhang, Lei</creator><creator>Li, Ninghuan</creator><creator>Zhu, Chencen</creator><creator>Chen, Junsheng</creator><creator>Luo, Han</creator><creator>Wang, Jiaxian</creator><creator>Feng, Lei</creator><creator>Lu, Huili</creator><creator>Zhu, Jianwei</creator><general>Springer Berlin Heidelberg</general><general>Springer Nature 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>3V.</scope><scope>7QL</scope><scope>7T7</scope><scope>7WY</scope><scope>7WZ</scope><scope>7X7</scope><scope>7XB</scope><scope>87Z</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8FL</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BEZIV</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FRNLG</scope><scope>FYUFA</scope><scope>F~G</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K60</scope><scope>K6~</scope><scope>K9.</scope><scope>L.-</scope><scope>LK8</scope><scope>M0C</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PQBIZ</scope><scope>PQBZA</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0002-8748-7899</orcidid></search><sort><creationdate>20170201</creationdate><title>A novel self-cleavable tag Zbasic–∆I-CM and its application in the soluble expression of recombinant human interleukin-15 in Escherichia coli</title><author>Shi, Siwei ; Chen, Huanhuan ; Jiang, Hua ; Xie, Yueqing ; Zhang, Lei ; Li, Ninghuan ; Zhu, Chencen ; Chen, Junsheng ; Luo, Han ; Wang, Jiaxian ; Feng, Lei ; Lu, Huili ; Zhu, Jianwei</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3242-18e39fc5c2ca69f04b2a67252cfc3d50a13edf2da0a4c6efcf914de2ed1463403</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2017</creationdate><topic>Analysis</topic><topic>Biomedical and Life Sciences</topic><topic>Biopharmaceutics - methods</topic><topic>Biotechnologically Relevant Enzymes and Proteins</topic><topic>Biotechnology</topic><topic>Chromatography</topic><topic>Chromatography, Liquid</topic><topic>Clinical trials</topic><topic>Cytokines</topic><topic>E coli</topic><topic>Enzymes</topic><topic>Escherichia coli - genetics</topic><topic>Escherichia coli - metabolism</topic><topic>Gene expression</topic><topic>Genetic recombination</topic><topic>Humans</topic><topic>Hydrogen-Ion Concentration</topic><topic>Inteins</topic><topic>Interleukin-15 - chemistry</topic><topic>Interleukin-15 - genetics</topic><topic>Interleukin-15 - isolation & purification</topic><topic>Life Sciences</topic><topic>Liquid chromatography</topic><topic>Mass Spectrometry</topic><topic>Microbial Genetics and Genomics</topic><topic>Microbiology</topic><topic>Mycobacterium tuberculosis - genetics</topic><topic>Pharmaceutical sciences</topic><topic>Plasmids</topic><topic>Proteins</topic><topic>Recombinant Fusion Proteins - chemistry</topic><topic>Recombinant Fusion Proteins - 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Academic</collection><jtitle>Applied microbiology and biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Shi, Siwei</au><au>Chen, Huanhuan</au><au>Jiang, Hua</au><au>Xie, Yueqing</au><au>Zhang, Lei</au><au>Li, Ninghuan</au><au>Zhu, Chencen</au><au>Chen, Junsheng</au><au>Luo, Han</au><au>Wang, Jiaxian</au><au>Feng, Lei</au><au>Lu, Huili</au><au>Zhu, Jianwei</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A novel self-cleavable tag Zbasic–∆I-CM and its application in the soluble expression of recombinant human interleukin-15 in Escherichia coli</atitle><jtitle>Applied microbiology and biotechnology</jtitle><stitle>Appl Microbiol Biotechnol</stitle><addtitle>Appl Microbiol Biotechnol</addtitle><date>2017-02-01</date><risdate>2017</risdate><volume>101</volume><issue>3</issue><spage>1133</spage><epage>1142</epage><pages>1133-1142</pages><issn>0175-7598</issn><eissn>1432-0614</eissn><abstract>Soluble expression of recombinant therapeutic proteins in
Escherichia coli
(
E. coli
) has been a challenging task in biopharmaceutical development. In this study, a novel self-cleavable tag Zbasic–intein has been constructed for the soluble expression and purification of a recombinant cytokine, human interleukin-15 (IL-15). We screened several solubilizing tags fused with the self-cleavable
Mycobacterium tuberculosis
recA mini-intein ∆I-CM and demonstrated that Zbasic tag can significantly improve the solubility of the product with correspondent to the intein activity. The fusion protein “Zbasic–∆I-CM–IL-15” was expressed with high solubility and easily enriched by the cost-effective cation-exchange chromatography. The self-cleavage of the fusion tag Zbasic–∆I-CM was then induced by a pH shift, with an activation energy of 7.48 kcal/mol. The mature IL-15 with natural N-terminus was released and further purified by hydrophobic interaction and anion-exchange chromatography. High-resolution reverse-phase high-performance liquid chromatography and mass spectrometry analysis confirmed that the product was of high purity and correct mass. With a CTLL-2 cell proliferation-based assay, the EC
50
was evaluated to be of about 0.126 ng/mL, similar to the product in clinical trials. By avoiding the time-consuming denaturing-refolding steps in previously reported processes, the current method is efficient and cost-effective. The novel tag Zbasic–∆I-CM can be potentially applied to large-scale manufacturing of recombinant human cytokines as well as other mammalian-sourced proteins in
E. coli
.</abstract><cop>Berlin/Heidelberg</cop><pub>Springer Berlin Heidelberg</pub><pmid>27683210</pmid><doi>10.1007/s00253-016-7848-2</doi><tpages>10</tpages><orcidid>https://orcid.org/0000-0002-8748-7899</orcidid></addata></record> |
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| ispartof | Applied microbiology and biotechnology, 2017-02, Vol.101 (3), p.1133-1142 |
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| language | eng |
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| source | MEDLINE; SpringerNature Journals |
| subjects | Analysis Biomedical and Life Sciences Biopharmaceutics - methods Biotechnologically Relevant Enzymes and Proteins Biotechnology Chromatography Chromatography, Liquid Clinical trials Cytokines E coli Enzymes Escherichia coli - genetics Escherichia coli - metabolism Gene expression Genetic recombination Humans Hydrogen-Ion Concentration Inteins Interleukin-15 - chemistry Interleukin-15 - genetics Interleukin-15 - isolation & purification Life Sciences Liquid chromatography Mass Spectrometry Microbial Genetics and Genomics Microbiology Mycobacterium tuberculosis - genetics Pharmaceutical sciences Plasmids Proteins Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - economics Recombinant Fusion Proteins - isolation & purification Solubility Studies |
| title | A novel self-cleavable tag Zbasic–∆I-CM and its application in the soluble expression of recombinant human interleukin-15 in Escherichia coli |
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