Study on Cecropin B2 Production via Construct Bearing Intein Oligopeptide Cleavage Variants
In this study, genetic engineering was applied to the overexpression of the antimicrobial peptide (AMP) cecropin B2 (cecB2). pTWIN1 vector with a chitin-binding domain (CBD) and an auto-cleavage Ssp DnaB intein (INT) was coupled to the cecB2 to form a fusion protein construct and expressed via <i...
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| Format: | Article |
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MDPI AG
2020-02-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/25/4/1005 |
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| author | Yi-Ting Fang Si-Yu Li Nien-Jen Hu Jie Yang Jyung-Hurng Liu Yung-Chuan Liu |
| author_facet | Yi-Ting Fang Si-Yu Li Nien-Jen Hu Jie Yang Jyung-Hurng Liu Yung-Chuan Liu |
| author_sort | Yi-Ting Fang |
| collection | DOAJ |
| description | In this study, genetic engineering was applied to the overexpression of the antimicrobial peptide (AMP) cecropin B2 (cecB2). pTWIN1 vector with a chitin-binding domain (CBD) and an auto-cleavage Ssp DnaB intein (INT) was coupled to the cecB2 to form a fusion protein construct and expressed via <i>Escherichia coli</i> ER2566. The cecB2 was obtained via the INT cleavage reaction, which was highly related to its adjacent amino acids. Three oligopeptide cleavage variants (OCVs), i.e., GRA, CRA, and SRA, were used as the inserts located at the C-terminus of the INT to facilitate the cleavage reaction. SRA showed the most efficient performance in accelerating the INT self-cleavage reaction. In addition, in order to treat the INT as a biocatalyst, a first-order rate equation was applied to fit the INT cleavage reaction. A possible inference was proposed for the INT cleavage promotion with varied OCVs using a molecular dynamics (MD) simulation. The production and purification via the CBD-INT-SRA-cecB2 fusion protein resulted in a cecB2 yield of 58.7 mg/L with antimicrobial activity. |
| first_indexed | 2024-12-22T11:44:38Z |
| format | Article |
| id | doaj.art-5688f9f908064ebf8b3e2fa3aca6d5e0 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-12-22T11:44:38Z |
| publishDate | 2020-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-5688f9f908064ebf8b3e2fa3aca6d5e02022-12-21T18:27:10ZengMDPI AGMolecules1420-30492020-02-01254100510.3390/molecules25041005molecules25041005Study on Cecropin B2 Production via Construct Bearing Intein Oligopeptide Cleavage VariantsYi-Ting Fang0Si-Yu Li1Nien-Jen Hu2Jie Yang3Jyung-Hurng Liu4Yung-Chuan Liu5Department of Chemical Engineering, National Chung Hsing University, Taichung 40227, TaiwanDepartment of Chemical Engineering, National Chung Hsing University, Taichung 40227, TaiwanGraduate Institute of Biochemistry, National Chung Hsing University, Taichung 40227, TaiwanGraduate Institute of Biochemistry, National Chung Hsing University, Taichung 40227, TaiwanInstitute of Genomics and Bioinformatics, NCHU, Taichung 40227, TaiwanDepartment of Chemical Engineering, National Chung Hsing University, Taichung 40227, TaiwanIn this study, genetic engineering was applied to the overexpression of the antimicrobial peptide (AMP) cecropin B2 (cecB2). pTWIN1 vector with a chitin-binding domain (CBD) and an auto-cleavage Ssp DnaB intein (INT) was coupled to the cecB2 to form a fusion protein construct and expressed via <i>Escherichia coli</i> ER2566. The cecB2 was obtained via the INT cleavage reaction, which was highly related to its adjacent amino acids. Three oligopeptide cleavage variants (OCVs), i.e., GRA, CRA, and SRA, were used as the inserts located at the C-terminus of the INT to facilitate the cleavage reaction. SRA showed the most efficient performance in accelerating the INT self-cleavage reaction. In addition, in order to treat the INT as a biocatalyst, a first-order rate equation was applied to fit the INT cleavage reaction. A possible inference was proposed for the INT cleavage promotion with varied OCVs using a molecular dynamics (MD) simulation. The production and purification via the CBD-INT-SRA-cecB2 fusion protein resulted in a cecB2 yield of 58.7 mg/L with antimicrobial activity.https://www.mdpi.com/1420-3049/25/4/1005chitin-binding domaininteinoligopeptide cleavage variantspurificationmolecular dynamics simulationfirst-order rate equation |
| spellingShingle | Yi-Ting Fang Si-Yu Li Nien-Jen Hu Jie Yang Jyung-Hurng Liu Yung-Chuan Liu Study on Cecropin B2 Production via Construct Bearing Intein Oligopeptide Cleavage Variants Molecules chitin-binding domain intein oligopeptide cleavage variants purification molecular dynamics simulation first-order rate equation |
| title | Study on Cecropin B2 Production via Construct Bearing Intein Oligopeptide Cleavage Variants |
| title_full | Study on Cecropin B2 Production via Construct Bearing Intein Oligopeptide Cleavage Variants |
| title_fullStr | Study on Cecropin B2 Production via Construct Bearing Intein Oligopeptide Cleavage Variants |
| title_full_unstemmed | Study on Cecropin B2 Production via Construct Bearing Intein Oligopeptide Cleavage Variants |
| title_short | Study on Cecropin B2 Production via Construct Bearing Intein Oligopeptide Cleavage Variants |
| title_sort | study on cecropin b2 production via construct bearing intein oligopeptide cleavage variants |
| topic | chitin-binding domain intein oligopeptide cleavage variants purification molecular dynamics simulation first-order rate equation |
| url | https://www.mdpi.com/1420-3049/25/4/1005 |
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