Immobilization and Purification of Enzymes With the Novel Affinity Tag ChBD-AB From Chitinolyticbacter meiyuanensis SYBC-H1
A new protein immobilization and purification system has been developed based on the improved plasmid vectors, designated pETChBD-X, which contained the gene coding for two novel chitin-binding domains ChBD-AB, factor Xa cleavage site and adapted for gene fusions. The ChBD-AD from Chitinolyticbacter...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2020-06-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fbioe.2020.00579/full |
| _version_ | 1828735028577697792 |
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| author | Jie Zhou Jianhao Chen Nisha Zhuang Alei Zhang Alei Zhang Kequan Chen Kequan Chen Ning Xu Fengxue Xin Fengxue Xin Wenming Zhang Wenming Zhang Weiliang Dong Weiliang Dong Min Jiang Min Jiang |
| author_facet | Jie Zhou Jianhao Chen Nisha Zhuang Alei Zhang Alei Zhang Kequan Chen Kequan Chen Ning Xu Fengxue Xin Fengxue Xin Wenming Zhang Wenming Zhang Weiliang Dong Weiliang Dong Min Jiang Min Jiang |
| author_sort | Jie Zhou |
| collection | DOAJ |
| description | A new protein immobilization and purification system has been developed based on the improved plasmid vectors, designated pETChBD-X, which contained the gene coding for two novel chitin-binding domains ChBD-AB, factor Xa cleavage site and adapted for gene fusions. The ChBD-AD from Chitinolyticbacter meiyuanensis SYBC-H1 was used as a novel affinity tag to anchor fusion proteins to chitin granules. The granules carrying the ChBD-AD fusion proteins can be isolated by a simple centrifugation step and used directly for some applications. Moreover, when required, a practically pure preparation of the soluble recombination protein can be obtained after Factor Xa cleavage. The efficiency of this system has been demonstrated by reaching 95% of protein absorbed to chitin within 30 min and recycling over 75% of interest protein after Factor Xa cleavage to separate interest protein and fusion tag. Furthermore, 65% L-glutamate oxidase with this fusion tag could be purified and immobilized within only one step and to be reused in converting L-glutamate to α-ketoglutaric acid directly, the average conversion rate kept above 65% even within four batches of enzyme conversion reaction. |
| first_indexed | 2024-04-12T22:58:09Z |
| format | Article |
| id | doaj.art-b857deec93d042ffbb817cc085601b78 |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-04-12T22:58:09Z |
| publishDate | 2020-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-b857deec93d042ffbb817cc085601b782022-12-22T03:13:07ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852020-06-01810.3389/fbioe.2020.00579544642Immobilization and Purification of Enzymes With the Novel Affinity Tag ChBD-AB From Chitinolyticbacter meiyuanensis SYBC-H1Jie Zhou0Jianhao Chen1Nisha Zhuang2Alei Zhang3Alei Zhang4Kequan Chen5Kequan Chen6Ning Xu7Fengxue Xin8Fengxue Xin9Wenming Zhang10Wenming Zhang11Weiliang Dong12Weiliang Dong13Min Jiang14Min Jiang15State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, ChinaState Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, ChinaState Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, ChinaState Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, ChinaJiangsu National Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, Nanjing, ChinaState Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, ChinaJiangsu National Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, Nanjing, ChinaState Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, ChinaState Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, ChinaJiangsu National Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, Nanjing, ChinaState Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, ChinaJiangsu National Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, Nanjing, ChinaState Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, ChinaJiangsu National Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, Nanjing, ChinaState Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, ChinaJiangsu National Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, Nanjing, ChinaA new protein immobilization and purification system has been developed based on the improved plasmid vectors, designated pETChBD-X, which contained the gene coding for two novel chitin-binding domains ChBD-AB, factor Xa cleavage site and adapted for gene fusions. The ChBD-AD from Chitinolyticbacter meiyuanensis SYBC-H1 was used as a novel affinity tag to anchor fusion proteins to chitin granules. The granules carrying the ChBD-AD fusion proteins can be isolated by a simple centrifugation step and used directly for some applications. Moreover, when required, a practically pure preparation of the soluble recombination protein can be obtained after Factor Xa cleavage. The efficiency of this system has been demonstrated by reaching 95% of protein absorbed to chitin within 30 min and recycling over 75% of interest protein after Factor Xa cleavage to separate interest protein and fusion tag. Furthermore, 65% L-glutamate oxidase with this fusion tag could be purified and immobilized within only one step and to be reused in converting L-glutamate to α-ketoglutaric acid directly, the average conversion rate kept above 65% even within four batches of enzyme conversion reaction.https://www.frontiersin.org/article/10.3389/fbioe.2020.00579/fullchitin-binding domainfusion tagaffinity chromatographyprotein purificationprotein immobilizationenzyme conversion |
| spellingShingle | Jie Zhou Jianhao Chen Nisha Zhuang Alei Zhang Alei Zhang Kequan Chen Kequan Chen Ning Xu Fengxue Xin Fengxue Xin Wenming Zhang Wenming Zhang Weiliang Dong Weiliang Dong Min Jiang Min Jiang Immobilization and Purification of Enzymes With the Novel Affinity Tag ChBD-AB From Chitinolyticbacter meiyuanensis SYBC-H1 Frontiers in Bioengineering and Biotechnology chitin-binding domain fusion tag affinity chromatography protein purification protein immobilization enzyme conversion |
| title | Immobilization and Purification of Enzymes With the Novel Affinity Tag ChBD-AB From Chitinolyticbacter meiyuanensis SYBC-H1 |
| title_full | Immobilization and Purification of Enzymes With the Novel Affinity Tag ChBD-AB From Chitinolyticbacter meiyuanensis SYBC-H1 |
| title_fullStr | Immobilization and Purification of Enzymes With the Novel Affinity Tag ChBD-AB From Chitinolyticbacter meiyuanensis SYBC-H1 |
| title_full_unstemmed | Immobilization and Purification of Enzymes With the Novel Affinity Tag ChBD-AB From Chitinolyticbacter meiyuanensis SYBC-H1 |
| title_short | Immobilization and Purification of Enzymes With the Novel Affinity Tag ChBD-AB From Chitinolyticbacter meiyuanensis SYBC-H1 |
| title_sort | immobilization and purification of enzymes with the novel affinity tag chbd ab from chitinolyticbacter meiyuanensis sybc h1 |
| topic | chitin-binding domain fusion tag affinity chromatography protein purification protein immobilization enzyme conversion |
| url | https://www.frontiersin.org/article/10.3389/fbioe.2020.00579/full |
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